Macrocyclic ligands and complexes

ABSTRACT

A group of functionalized polyaminocarboxylate chelants that form complexes with rare earth-type metal ions are disclosed. The complexes, covalently attached to an antibody or antibody fragment, can be used for therapeutic and/or diagnostic purposes.

CROSS-REFERENCE TO A RELATED APPLICATION

This is a divisional of application Ser. No. 07,962,168 filed Oct. 15,1992 now U.S. Pat. No. 5,435,990 which application is acontinuation-in-part application of U.S. application Ser. No. 211,496,filed Jun. 24, 1988 now abandoned.

BACKGROUND OF THE INVENTION

Functionalized chelants, or bifunctional coordinators, are known to becapable of being covalently attached to an antibody having specificityfor cancer or tumor cell epitopes or antigens. Radionuclide complexes ofsuch antibody/chelant conjugates are useful in diagnostic and/ortherapeutic applications as a means of conveying the radionuclide to acancer or tumor cell. See, for example, Meares et al., Anal. Biochem.142, 68-78, (1984); and Krejcarek et al., Biochem. and Biophys. Res.Comm. 77, 581-585 (1977).

Aminocarboxylic acid chelating agents have been known and studied formany years. Typical of the aminocarboxylic acids are nitrilotriaceticacid (NTA), ethylenediaminetetraacetic acid (EDTA),hydroxyethylethylenediaminetriacetic acid (HEDTA),diethylenetriaminepentaacetic acid (DTPA),trans-1,2-diaminocyclohexanetetraacetic acid (CDTA) and1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA). Numerousbifunctional chelating agents based on aminocarboxylic acids have beenproposed and prepared. For example the cyclic dianhydride of DTPA[Hnatowich et al. Science 220, 613-615, (1983); U.S. Pat. No. 4,479,930]and mixed carboxycarbonic anhydrides of DTPA [Gansow, U.S. Pat. Nos.4,454,106 and 4,472,509; Krejcarek et al., Biochem. and Biophys. Res.Comm. 77, 581-585, (1977)] have been reported. When the anhydrides arecoupled to proteins the coupling proceeds via formation of an amide bondthus leaving four of the original five carboxymethyl groups on thediethylenetriamine (DETA) backbone [Hnatowich et al. Int. J. Appl. Isot.33, 327-332, (1982)]. In addition, U.S. Pat. Nos. 4,432,907 and4,352,751 disclose bifunctional chelating agents useful for bindingmetal ions to "organic species such as organic target molecules orantibodies." As in the above, coupling is obtained via an amide groupthrough the utilization of diaminotetraacetic acid dianhydrides.Examples of anhydrides include dianhydrides of EDTA, CDTA,propylenediaminetetraacetic acid and phenylene 1,2-diaminetetraaceticacid. A recent U.S. Pat. No. 4,647,447 discloses several complex saltsformed from the anion of a complexing acid for use in various diagnostictechniques. Conjugation via a carboxyl group of the complexing acid istaught which gives a linkage through an amide bond.

In the J. of Radioanalytical Chemistry 57(12), 553-564 (1980), Paik etal. disclose the use of p-nitrobenzylbromide in a reaction with a"blocked" diethylenetriamine, i.e. bis-(2-phthalimidoethyl)aminefollowed by deblocking procedures and carboxymethylation usingchloroacetic acid, to give N'-p-nitrobenzyldiethylenetriamineN,N,N",N"-tetraacetic acid. Again, since the attachment is through anitrogen, a tetraacetic acid derivative is obtained. Conjugation of thebifunctional chelating agent and chelation with indium is discussed.Substitution on the nitrogen atom is also taught by Eckelman, et al. inthe J. of Pharm. Sci. 64(4), 704-706 (1975) by reacting amines such as"ethylenediamine or diethylenetriamine with the appropriate alkylbromide before carboxymethylation." The compounds are proposed aspotential radiopharmaceutical imaging agents.

Another class of bifunctional chelating agents based on aminocarboxylicacid functionality is also well documented in the literature. Thus,Sundberg, Meares, et al. in the J. of Med. Chem. 17(12), 1304 (1974),disclosed bifunctional analogs of EDTA. Representative of thesecompounds are 1-(p-aminophenyl)-ethylenediaminetetraacetic acid and1-(p-benzene-diazonium)-ethylenediaminetetraacetic acid. Coupling toproteins through the para-substituent and the binding of radioactivemetal ions to the chelating group is discussed. The compounds are alsodisclosed in Biochemical and Biophysical Research Communications 75(1),149 (1977), and in U.S. Pat. Nos. 3,994,966 and 4,043,998. It isimportant to note that the attachment of the aromatic group to the EDTAstructure is through a carbon of the ethylenediamine backbone. Opticallyactive bifunctional chelating agents based on EDTA, HEDTA and DTPA aredisclosed in U.S. Pat. No. 4,622,420. In these compounds an alkylenegroup links the aromatic group (which contains the functionality neededfor attachment to the protein) to the carbon of the polyamine whichcontains the chelating functionality. Other references to such compoundsinclude Brechbiel et al., Inorg. Chem. 25, 2772-2781 (1986), U.S. Pat.No. 4,647,447 and International Pat. Publication No. WO 86/06384.

More recently, certain macrocyclic bifunctional chelating agents and theuse of their copper chelate conjugates for diagnostic or therapeuticapplications have been disclosed in U.S. Pat. No. 4,678,667 and by Moiet al., Inorg. Chem. 26, 3458-3463 (1987). Attachment of theaminocarboxylic acid functionality to the rest of the bifunctionalchelating molecule is through a ring carbon of the cyclic polyaminebackbone. Thus, a linker, attached at one end to a ring carbon of thecyclic polyamine, is also attached at its other end to a functionalgroup capable of reacting with the protein.

Another class of bifunctional chelating agents, also worthy of note,consists of compounds wherein the chelating moiety, i.e. theaminocarboxylic acid, of the molecule is attached through a nitrogen tothe functional group of the molecule containing the moiety capable ofreacting with the protein. As an example, Mikola et al. in patentapplication (International Publication No. WO 84/03698, published Sep.27, 1984) disclose a bifunctional chelating agent prepared by reactingp-nitrobenzylbromide with DETA followed by reaction with bromoaceticacid to make the aminocarboxylic acid. The nitro group is reduced to thecorresponding amine group and is then converted to the isothiocyanategroup by reaction with thiophosgene. These compounds are bifunctionalchelating agents capable of chelating lanthanides which can beconjugated to bioorganic molecules for use as diagnostic agents. Sinceattachment of the linker portion of the molecule is through one of thenitrogens of the aminocarboxylic acid, then one potential aminocarboxylgroup is lost for chelation. Thus, a DETA-based bifunctional chelantcontaining four (not five) acid groups is prepared. In this respect,this class of bifunctional chelant is similar to those where attachmentto the protein is through an amide group with subsequent loss of acarboxyl chelating group.

Recently Carney, Rogers, and Johnson disclosed (3rd. InternationalConference on Monoclonal Antibodies For Cancer; San Diego, Calif.-Feb.4-6, 1988) abstracts entitled "Absence of Intrinsically Higher TissueUptake from Indium-111 Labeled Antibodies: Co-administration ofIndium-111 and Iodine-125 Labeled B72.3 in a Nude Mouse Model" and"Influence of Chelator Denticity on the Biodistribution of Indium-111Labeled B72.3 Immuno-conjugates in Nude Mice". The biodistribution ofindium-111 complexed with an EDTA and DTPA bifunctional chelating agentis disclosed. Attachment of the aromatic ring to the EDTA/DTPA moietiesis through an acetate methylene. Also at a recent meeting D. K. Johnsonet al. [Florida Conf. on Chem. in Biotechnology, Apr. 26-29 (1988), PalmCoast, Fla.] disclosed bifunctional derivatives of EDTA and DTPA where ap-isothiocyanatobenzyl moiety is attached at the methylene carbon of oneof the carboxymethyl groups. Previously Hunt et al. in U.S. Pat. Nos.4,088,747 and 4,091,088 (1978) disclosed ethylenediaminediacetic acid(EDDA) based chelating agents wherein attachment of an aromatic ring tothe EDDA moiety is through the alkylene or acetate methylene. Thecompounds are taught to be useful as chelates for studying hepatobiliaryfunction. The preferred metal is technetium-99m. Indium-111 andindium-113m are also taught as useful radionuclides for imaging.

Consequently, it would be advantageous to provide a complex that doesnot readily dissociate, that exhibits rapid whole body clearance exceptfrom the desired tissue, and conjugates with an antibody to produce thedesired results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 and 15-21 show the biodistribution of ¹⁵³ Sm as administeredas a conjugate containing ¹⁵³ Sm of the present invention. The antibody,CC₄₉ -IgG, was used in the conjugate of the present invention. Thebiodistribution was determined in nude mice bearing LS 174-T tumor.

FIGS. 8-14 and 22-28 show the biodistribution of ¹⁵³ Sm as administeredas a conjugate containing ¹⁵³ Sm of the present invention. The conjugateof the present invention used CC₄₉ -F(ab')₂ as the antibody fragment.The biodistribution was determined in nude mice bearing LS 174-T tumor.

FIGS. 29-34 show the biodistribution of ¹⁷⁷ Lu as a conjugate containing¹⁷⁷ Lu(PA-DOTMA) or ¹⁷⁷ Lu(PA-DOTA). The conjugate used CC₄₉ -IgG as theantibody. The biodistribution was determined in (balb/C) mice bearing LS174-T tumor.

SUMMARY OF THE INVENTION

Surprisingly, the complexes and/or conjugates of the invention arerelatively stable (i.e. do not easily dissociate) and some display rapidclearance from the whole body and some non-target organs, such as liver,kidney and bone.

The invention includes the design and synthesis of novel bifunctionalchelants, each containing a chelating functionality, and a chemicallyreactive group for covalent attachment to biomolecules. Also formingpart of the invention are methods for preparing various bifunctionalcoordinator (BFC)-metal complexes and the linking of the complexes toantibody to prepare radionuclide (such as samarium-153, lutetium-177 andyttrium-90) labeled antibody and/or fragments suitable for diagnosticand/or therapeutic applications.

The present invention is directed to novel bifunctional chelating agentsthat form complexes with metal ions, especially "radioactive" metal ionshaving rare earth-type chemistry. Preferred rare earth-type metal ionsinclude La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Yand Sc, especially preferred are Sm, Ho, Y and Lu. Preferred radioactiverare earth-type metal ions include ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ¹⁴⁹ Pm, ¹⁵⁹ Gd,¹⁴⁰ La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ Sc, and ¹⁴² Pr, especially preferred are ¹⁵³Sm, ¹⁶⁶ Ho, ⁹⁰ Y and ¹⁷⁷ Lu. Other radioactive metal ions which may beof interest are ⁴⁷ Sc, ^(99m) Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd,¹⁹⁷ Pt, ⁶⁷ Cu, ¹⁹⁸ Au, ¹⁹⁹ Au, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ^(113m) In, ^(115m)In, ^(117m) Sn, and ²¹² Pb/²¹² Bi. The complexes so formed can beattached (covalently bonded) to an antibody or fragment thereof and usedfor therapeutic and/or diagnostic purposes. The complexes and/orconjugates can be formulated for in vivo or in vitro uses. A preferreduse of the formulated conjugates is the treatment of cancer in animals,especially humans.

Uses of the complexes and/or conjugates of this invention which containa non-radioactive metal for diagnosis and/or treatment of disease statessuch as cancer are also possible. Such uses are known fornon-radioactive metals using radio frequency to induce hyperthermia(Jpn. Kokai Tokkyo Koho JP 61,158,931) and fluorescent-immunoguidedtherapy (FIGS) [K. Pettersson et al., Clinical Chemistry 29 (1), 60-64(1983) and C. Meares et al., Acc. Chem. Res. 17, 202-209 (1984].

More specifically, the present invention is directed to a compound ofthe formula: ##STR1## wherein: each Q is independently hydrogen or(CHR^(5'))_(p) CO₂ R;

Q¹ is hydrogen or (CHR^(5'))_(w) CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least two of the sum of Q and Q¹ must be otherthan hydrogen;

each R^(5') independently is hydrogen, C₁ -C₄ alkyl or --(C₁ -C₂alkyl)phenyl;

X and Y are each independently hydrogen or may be taken with an adjacentX and Y to form an additional carbon-carbon bond;

n is 0 or 1;

m is an integer from 0 to 10 inclusive;

p=1 or 2;

r=0 or 1;

w=0 or 1;

with the proviso that n is only 1 when X and/or Y form an additionalcarbon-carbon bond, and the sum of r and w is 0 or 1;

L is a linker/spacer group covalently bonded to, and replaces onehydrogen atom of one of the carbon atoms to which it is joined, saidlinker/spacer group being represented by the formula ##STR2## wherein: sis an integer of 0 or 1;

t is an integer of 0 to 20 inclusive;

R¹ is an electrophilic or nucleophilic moiety which allows for covalentattachment to an antibody or fragment thereof, or a synthetic linkerwhich can be attached to an antibody or fragment thereof, or precursorthereof; and

Cyc represents a cyclic aliphatic moiety, aromatic moiety, aliphaticheterocyclic moiety, or aromatic heterocyclic moiety, each of saidmoieties optionally substituted with one or more groups which do notinterfere with binding to an antibody or antibody fragment;

with the proviso that when s, t, m, r and n are 0, then R¹ is other thancarboxyl; or

pharmaceutically acceptable salt thereof.

Preferred features of the compounds of formula (I) are those where: R ishydrogen; R^(5') is H or methyl; n is 0; m is 0 through 5; r is 0; and Lis a compound of the formula: ##STR3## wherein: R² is selected from thegroup consisting of hydrogen, nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido andmaleimido;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen;

R⁴ is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido;

with the proviso that R² and R⁴ cannot both be hydrogen but one of R²and R⁴ must be hydrogen; or

a pharmaceutically acceptable salt thereof.

When a conjugate of the present invention is desired R² and R⁴ must beother than nitro. When R² or R⁴ is nitro, then a precursor of the linkermoiety (L) is present. This precursor moiety can be any moiety which isformed for R² or R⁴ for the preparation of the compounds of formula (I)and which does not bind to an antibody or antibody fragment.

The present invention is also directed to rare-earth type metal ioncomplexes, especially radioactive neutral or charged rare-earth typemetal ion complexes, and to conjugates formed with the aforementionedcomplexes and antibody or antibody fragments. In addition the presentinvention also includes formulations having the conjugates of theinvention and a pharmaceutically acceptable carrier, especiallyformulations where the pharmaceutically acceptable carrier is a liquid.The invention also includes a method for the diagnosis or treatment of adisease state, especially cancer, in a mammal which comprisesadministering to the mammal an effective amount of the formulation.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following indicated terms have these meanings: withrespect to the definition of R¹, R² or R⁴, "electrophilic" moietiesinclude, but are not limited to, isothiocyanate, bromoacetamide,maleimide, imidoester, thiophthalimide, N-hydroxysuccinimyl ester,pyridyl disulfide, and phenyl azide; suitable "nucleophilic" moietiesinclude, but are not limited to, carboxyl, amino, acyl hydrazide,semicarbazide, and thiosemicarbazide; "synthetic linkers" include anysynthetic organic or inorganic linkers which are capable of beingcovalently attached to an antibody or antibody fragment, preferredsynthetic linkers are biodegradable synthetic linkers which are stablein the serum of a patient but which have a potential for cleavage withinan organ of clearance for the radio-conjugate, for example biodegradablepeptides or peptide containing groups. Of the electrophilic moietiesisothiocyanate is preferred and of the nucleophilic moieties amino,semicarbazide and thiosemicarbazide are preferred. It is desirable thatthe nature and/or position of R¹ be such that it does not appreciablyinterfere with the chelation reaction.

The "X--C--Y" term in formula (I) represents the optional presence of adouble or triple bond between adjacent carbon atoms. The unsaturatedbonds may be present independently in the chain length of 0 to 10 carbonatoms inclusive as defined by the "m" term in formula (I).

As used herein, the term "mammal" means animals that nourish their youngwith milk secreted by mammary glands, preferably warm blooded mammals,more preferably humans. "Antibody" refers to any polyclonal, monoclonal,chimeric antibody or heteroantibody, preferably a monoclonal antibody;"antibody fragment" includes Fab fragments and F(ab')₂ fragments, andany portion of an antibody having specificity toward a desired epitopeor epitopes. When using the term "metal chelate/antibody conjugate" or"conjugate", the "antibody" portion is meant to include whole antibodiesand/or antibody fragments, including semisynthetic or geneticallyengineered variants thereof.

As used herein, "complex" refers to a compound of the invention, e.g.formula (I), complexed with a rare-earth type metal ion, especially aradioactive rare-earth type metal ion, wherein at least one metal atomis chelated or sequestered; "radioactive metal ion chelate/antibodyconjugate" or "radioactive metal ion conjugate" refers to a radioactivemetal ion conjugate that is covalently attached to an antibody orantibody fragment; "radioactive" when used in conjunction with the word"metal ion" refers to one or more isotopes of the rare-earth typeelements that emit particles and/or photons, such as ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰Y, ¹⁴⁹ Pm, ¹⁵⁹ Gd, ¹⁴⁰ La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ Sc, and ¹⁴² Pr; the terms"bifunctional coordinator", "bifunctional chelating agent" and"functionalized chelant" are used interchangeably and refer to compoundsthat have a chelant moiety capable of chelating a metal ion and alinker/spacer moiety covalently bonded to the chelant moiety that iscapable of serving as a means to covalently attach to an antibody orantibody fragment.

As used herein, "pharmaceutically acceptable salt" means any salt of acompound of formula (I) which is sufficiently non-toxic to be useful intherapy or diagnosis of mammals. Thus, the salts are useful inaccordance with this invention. Representative of those salts, which areformed by standard reactions, from both organic and inorganic sourcesinclude, for example, sulfuric, hydrochloric, phosphoric, acetic,succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic,mucic, glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric,formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic,sorbic, picric, benzoic, cinnamic acids and other suitable acids. Alsoincluded are salts formed by standard reactions from both organic andinorganic sources such as ammonium, alkali metal ions, alkaline earthmetal ions, and other similar ions.

Particularly preferred are the salts of the compounds of formula (I)where the salt is potassium, sodium, ammonium, or mixtures thereof.

Of course, the free acid of the compounds of formula (I) may be used,also the protonated form of the compounds, for example when thecarboxylate is protonated and/or the nitrogen atoms i.e. when the HClsalt is formed.

Preferred compounds of formula (I) include those compounds where Q¹ ishydrogen and L is represented by formula A as shown by the followingformula: ##STR4## wherein: each Q is independently hydrogen or CHR⁵ CO₂R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least two of Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R² is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen;

R⁴ is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl;

with the proviso that R² and R⁴ cannot both be hydrogen but one of R²and R⁴ must be hydrogen; or

a pharmaceutically acceptable salt thereof.

In formula (II) when R³ and R⁴ are both hydrogen, the compounds arerepresented by the following formula: ##STR5## wherein: each Q isindependently hydrogen or CHR⁵ CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least two of Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R² is selected from the group consisting of nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl; or

a pharmaceutically acceptable salt thereof.

In formula (II) when R² is hydrogen, the compounds are represented bythe formula: ##STR6## wherein: each Q is independently hydrogen or CHR⁵CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least two of Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ Hand hydroxy;

R⁴ is selected from the group consisting of nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido, and maleimido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl; or

a pharmaceutically acceptable salt thereof.

Additional preferred compounds of formula (I) include those compoundswhere at least one Q is hydrogen and are represented by the formula:##STR7## wherein: each Q is independently hydrogen or CHR⁵ CO₂ R;

Q¹ is hydrogen or (CHR⁵)_(w) CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least two of the sum of Q and Q¹ must be otherthan hydrogen and one of Q is hydrogen;

m is an integer from 0 to 5 inclusive;

w is 0 or 1;

R² is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimidoand bromoacetamido;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen;

R⁴ is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimidoand bromoacetamido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl;

with the proviso that R² and R⁴ cannot both be hydrogen but one of R²and R⁴ must be hydrogen; or

a pharmaceutically acceptable salt thereof.

Other preferred compounds of formula (I) include compounds where Q¹ isCO₂ R (w=o) and are represented by the formula: ##STR8## wherein: each Qis independently hydrogen or CHR⁵ CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least one Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R² is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimidoand bromoacetamido;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen;

R⁴ is selected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimidoand bromoacetamido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl;

with the proviso that R² and R⁴ cannot both be hydrogen but one of R²and R⁴ must be hydrogen; or

a pharmaceutically acceptable salt thereof.

Some preferred compounds of formula (VI) are those where R³ and R⁴ areboth hydrogen, the compounds are represented by the formula: ##STR9##wherein: each Q is independently hydrogen or CHR⁵ CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least one Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R² is selected from the group consisting of nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl,bromoacetamido and maleimido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl; or

a pharmaceutically acceptable salt thereof.

Other preferred compounds of formula (VI) are those where R² is hydrogenand are represented by the formula: ##STR10## wherein: each Q isindependently hydrogen or CHR⁵ CO₂ R;

each R independently is hydrogen, benzyl or C₁ -C₄ alkyl;

with the proviso that at least one Q must be other than hydrogen;

m is an integer from 0 to 5 inclusive;

R³ is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,and hydroxy;

R⁴ is selected from the group consisting of nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido,and bromoacetamido;

each R⁵ independently is hydrogen or C₁ -C₄ alkyl; or

a pharmaceutically acceptable salt thereof.

The bifunctional chelating agents described herein [represented by anyone of formulas (I)-(VIII)] can be used to chelate or sequester therare-earth type metal ions, particularly radioactive rare-earth typemetal ions, so as to form metal ion chelates (also referred to herein as"complexes"). The complexes, because of the presence of thefunctionalizing moiety [represented by "R¹ " in formula (I)], can beattached to functionalized supports, such as functionalized polymericsupports, or preferably, when a complex of formula (I) where Lrepresents formula (A) and R² and R⁴ must be other than nitro, can becovalently attached to proteins or more specifically to antibodies orantibody fragments. Thus the complexes described herein (represented byany one of formulas I-VIII complexed with rare-earth type metal ions,particularly radioactive rare-earth type metal ions) may be covalentlyattached to an antibody or antibody fragment and are referred to hereinas "conjugates".

The antibodies or antibody fragments which may be used in the conjugatesdescribed herein can be prepared by techniques well known in the art.Highly specific monoclonal antibodies can be produced by hybridizationtechniques well known in the art, see for example, Kohler and Milstein[Nature 256, 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976)].Such antibodies normally have a highly specific reactivity. In theradioactive metal ion conjugates, antibodies directed against anydesired antigen or hapten may be used. Preferably the antibodies whichare used in the radioactive metal ion conjugates are monoclonalantibodies, or fragments thereof having high specificity for a desiredepitope(s). Antibodies used in the present invention may be directedagainst, for example, tumors, bacteria, fungi, viruses, parasites,mycoplasma, differentiation and other cell membrane antigens, pathogensurface antigens, toxins, enzymes, allergens, drugs and any biologicallyactive molecules. Some examples of antibodies or antibody fragments areCC-11, CC-46, CC-49, CC-49 F(ab')₂, CC-83, CC-83 F(ab')₂, and B72.3.[See D. Colcher et al., Cancer Res. 48, 4597-4603 (Aug. 15, 1988) forCC-49, CC-83 and B72.3 antibodies. The hybridoma cell line B72.3 isdeposited in the American Type Culture Collection (ATCC) having theaccession number HB 8108. The various CC antibodies are disclosed inU.S. patent application Ser. No. 7-073,685, filed Jul. 15, 1987, whichis available through NTIS. The other murine monoclonal antibodies bindto epitopes of TAG-72, a tumor associated antigen.] A more complete listof antigens can be found in U.S. Pat. No. 4,193,983, which isincorporated herein by reference. The radioactive metal ion conjugatesof the present invention are particularly preferred for the diagnosisand treatment of various cancers.

The preferred rare-earth type (lanthanide or pseudo-lanthanide)complexes of the present invention are represented by the formula:

    C[Ln(BFC)]                                                 (IX)

wherein: Ln is wherein: Ln is a rare-earth metal (lanthanide) ion, suchas Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺,Tm³⁺, Yb³⁺ and Lu³⁺, or pseudo-lanthanide metal ion, such as Sc³⁺, Y³⁺and La³⁺ such as, especially preferred metal ions are Y³⁺, Ho³⁺, Lu³⁺ orSm³⁺ ; BFC represents a bifunctional chelant; and C represents apharmaceutically acceptable ion or group of ions of sufficient charge torender the entire complex neutral. If the BFC contains four or morenegatively charged moieties, then C is a cation or group of cations suchas H⁺, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, NH₄ ⁺, N(CH₃)₄ ⁺,N(C₂ H₅)₄ ⁺, N(C₃ H₇)₄ ⁺, N(C₄ H₉)₄ ⁺, As(C₆ H₅)₄ ⁺, [(C₆ H₅)₃ P=]₂ N⁺and other protonated amines. If the BFC contains three negativelycharged moieties, then C is not required. If the BFC contains twonegatively charged moieties, then C is an anion such as F⁻, Cl⁻, Br⁻,I⁻, ClO₄ ⁻, BF₄ ⁻, H₂ PO₄ ⁻, HCO₃ ⁻, HCO₂ ⁻, CH₃ SO₃ ⁻, H₃ C--C₆ H₄--SO₃ ⁻, PF₆ ⁻, CH₃ CO₂ ⁻ and B(C₆ H₅)₄ ⁻.

The conjugates of this invention, and in some instances the complexes ofthis invention, may be employed as a formulation. The formulationcomprises a compound of formula (I) with the antibody and/or metal ionand a physiologically acceptable carrier, excipient or vehicletherefore. Thus, the formulation may consist of a physiologicallyacceptable carrier with a complex (metal ion+ligand), conjugate (metalion+ligand+antibody) or (ligand+antibody). The methods for preparingsuch formulations are well known. The formulation may be in the form ofa suspension, injectable solution or other suitable formulation.Physiologically acceptable suspending media, with or without adjuvants,may be used.

The formulations of the present invention are in the solid or liquidform containing the active radionuclide complexed with the ligand. Theseformulations may be in kit form such that the two components (i.e.ligand and metal, complex and antibody, or ligand/antibody and metal)are mixed at the appropriate time prior to use. Whether premixed or as akit, the formulations usually require a pharmaceutically acceptablecarrier.

Injectable compositions of the present invention may be either insuspension or solution form. In the preparation of suitable formulationsit will be recognized that, in general, the water solubility of the saltis greater than the acid form. In solution form the complex (or whendesired the separate components) is dissolved in a physiologicallyacceptable carrier. Such carriers comprise a suitable solvent,preservatives such as benzyl alcohol, if needed, and buffers. Usefulsolvents include, for example, water, aqueous alcohols, glycols, andphosphonate or carbonate esters. Such aqueous solutions contain no morethan 50 percent of the organic solvent by volume.

Injectable suspensions are compositions of the present invention thatrequire a liquid suspending medium, with or without adjuvants, as acarrier. The suspending medium can be, for example, aqueouspolyvinylpyrrolidone, inert oils such as vegetable oils or highlyrefined mineral oils, or aqueous carboxymethylcellulose. Suitablephysiologically acceptable adjuvants, if necessary to keep the complexin suspension, may be chosen from among thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin, and thealginates. Many surfactants are also useful as suspending agents, forexample, lecithin, alkylphenol, polyethylene oxide adducts,napthalenesulfonates, alkylbenzenesulfonates, and the polyoxyethylenesorbitan esters.

Many substances which effect the hydrophilicity, density, and surfacetension of the liquid suspension medium can assist in making injectablesuspensions in individual cases. For example, silicone antifoames,sorbitol, and sugars are all useful suspending agents.

An "effective amount" of the formulation is used for therapy. The dosewill vary depending on the disease being treated. Although in vitrodiagnostics can be performed with the formulations of this invention, invivo diagnostics are also contemplated using formulations of thisinvention. The conjugates and formulations of this invention can also beused in radioimmuno guided surgery (RIGS); however, other metals whichcould be used for this purpose also include ^(99m) Tc, ¹¹¹ In, ^(113m)In, ⁶⁷ Ga and ⁶⁸ Ga.

Other uses of some of the chelants of the present invention may includemagnetic resonance imaging, e.g. the complexes of formula I, especiallycomplexes of formula VI, with Gd⁺³, attachment to polymeric supports forvarious purposes, e.g. as diagnostic agents, and removal of lanthanidemetal or pseudo-lanthanide metal ion by selective extraction.

The present invention provides chelants, complexes, and antibodyconjugates some of which are more stable, and/or have improvedbiodistribution, and/or have more rapid clearance from the body, thanthose known in the art.

DETAILED DESCRIPTION OF THE PROCESS

A reasonable and general synthetic approach to a twelve-memberedmacrocyclic, bifunctional chelant of the present invention asrepresented by formula (I) involves monofunctionalization of a free-basemacrocycle (e.g. 1,4,7,10-tetraazacyclododecane) at only one of thenitrogen atoms with an appropriate electrophile (e.g. any appropriatelysubstituted α-halocarboxylic acid ester). This electrophile must possessa suitable linker moiety or suitably protected linker moiety which wouldallow covalent attachment of the bifunctional ligand to a protein,antibody or antibody fragment.

It is recognized in the art that alkylative techniques for theproduction of mono-N-functional polyazamacrocycles may result inmixtures of difficult to separate products [T. Kaden, Top. Curr. Chem.121, 157-75 (1984)]. This problem has been overcome through the reporteduse of large excesses of macrocycle (5-10 equivalents relative toelectrophile) which favors formation of the monoalkylation adduct [M.Studer and T. A. Kaden, Helv. Chim. Acta. 69, 2081-86 (1986); E. Kimuraet al., J. Chem. Soc. Chem. Commun. 1158-59 (1986)].

Other routes to mono-N-functional polyazamacrocycles involve lengthyprotection, functionalization, and deprotection schemes [P. S.Pallavicini et al., J. Amer. Chem. Soc. 109, 5139-44 (1987); M. F.Tweedle et al., Eur. Pub. Pat. Appln. No. 0232-751]. A recent report ofa reductive amination of substituted phenyl pyruvic acids and amines hasissued by Abbott Labs. as an Abstract from a recent meeting (D. K.Johnson et al., Florida Conf. on Chem. in Biotechnology, Apr. 26-29(1988), Palm Coast, Fla.). A process that prepares mono-N-alkylatedpolyazamacrocycles by using an electrophile with between about one andfive equivalents of a suitable macrocycle in a solvent which will notpromote a proton transfer is disclosed in U.S. application Ser. No.289,163, by W. J. Kruper, filed Dec. 22, 1988, the disclosure of whichis hereby incorporated by reference.

General synthetic routes to the chelants of the present invention aredisclosed in Synthesis Schemes I-IV hereinafter and involve the reactionof a suitable electrophile and the polyazamacrocycle, at variousstoichiometries, temperatures and concentrations, in an appropriateorganic solvent. Examples of suitable organic solvents are anyhydrocarbon which supports solubility, such as acetonitrile,isopropanol, methylene chloride, toluene, chloroform, n-butanol, carbontetrachloride, tetrahydrofuran, 5% ethanol in chloroform, with the mostpreferred being chloroform, methylene chloride, n-butanol, 1,4-dioxaneand acetonitrile. Stoichiometric amounts or nearly stoichiometricamounts of macrocycle and electrophile may be employed and yield thecorresponding mono-N-alkylation product in a single step. Thetemperature range for the reaction is from about 0° C. to about reflux,preferably from 0° C. to 25° C. The time of the reaction untilcompletion is about 1 to about 24 hours.

General access to substituted a-haloacid esters is desirable for theoverall viability of Synthesis Schemes II and IV. One suitable approachinvolves bromination or chlorination of the acid halide generated insitu, e.g. D.N. Harpp et al., J. Org. Chem. 40, 3420-27 (1975). Thisapproach allows for exclusive alpha halogenation of alkanoic acids whichcontain even reactive benzylic groups. A general method to substitutedacid halides involves reaction of the organic acid with thionyl chlorideor sulfuryl chloride, e.g. E. Schwenk et al. J. Amer. Chem. Soc. 70,3626-27 (1944). Both methods utilize the free carboxylic acid which isfrequently available from commercial sources.

Polyazamacrocycles such as 1,4,7,10-tetraazacyclododecane may beprepared by documented methods such as T. J. Atkins et al., J. Amer.Chem. Soc. 96, 2268-70 (1974) and T. J. Richman et al., Org. Synthesis58, 86-98 (1978).

Carboxymethylation of the mono-N-functional macrocycle may be performedby the method of Desreux using bromoacetic acid derivatives and asuitable base [J. F. Desreux, Inorg. Chem. 19, 1319-24 (1980)].

All of the starting materials required for preparing the compounds ofthis invention are either available from commercial sources or can bemade from known literature reference descriptions.

In the following Scheme I, the compounds of formula (I) are preparedwhere Q¹ is hydrogen. Although only one compound is indicated by theterms shown, other similar moieties within formula (I) where Q¹ ishydrogen, r=0 or 1, n=0 or 1, and m=0 through 10 can also be prepared bythis method. ##STR11##

In the following Scheme II, the compounds of formula (I) are preparedwhere Q¹ is hydrogen. Although only one compound is indicated by theterms shown, other similar moieties within formula (I) where Q¹ ishydrogen, r=0 or 1, n=0 or 1 and m=0 through 10 can also be prepared bythis method. ##STR12##

In the following Scheme III, the compounds of formula (I) are preparedwhere Q¹ is CO₂ R. Although only one compound is indicated by the termsshown, other similar moieties within formula (I) where Q¹ is CO₂ R,including m=0 through 10, n=0 or 1 and r=0 can also be prepared by thismethod. ##STR13##

In the following Scheme IV, the compounds of formula (I) are preparedwhere Q¹ is (CHR^(5'))_(w) CO₂ R. Although only one compound isindicated by the terms shown, other similar moieties within formula (I)where Q¹ is (CHR^(5'))_(w) CO₂ R, including m=0 through 10, n=0 or 1 andr=0 can also be prepared by this method.

In Scheme IV, the reagent shown containing A' can be any suitableleaving group for a α-acid, i.e. A' is phenyl or CF₃.

Use of an optically active alkylating agent has minimized thediastereomers and simplifies the synthesis. Isolation of a singlediastereomer which would give rise to a single, easily purifiedlanthanide complex would be desirable for radiochelation as well asantibody conjugation. ##STR14##

In the following Scheme V, the compounds of formula (I) are preparedwhere Q¹ is hydrogen or (CHR⁵)_(w) CO₂ R and R² is hydrogen. Althoughonly two compounds are indicated by the terms shown, other similarmoieties within formula (I) where Q¹ is hydrogen or (CHR⁵)_(w) CO₂ R, Qis hydrogen or (CHR⁵)_(p) CO₂ R, r=0 or 1, n=0 or 1, w=0 or 1, m=0through 10, p=1 or 2, L represents formula A wherein R³ is defined asbefore, R² and R⁴ are selected from the group consisting of hydrogen,amino, nitro, isothiocyanato, semicarbazido, thiosemicarbazido,maleimido, bromoacetamido and carboxyl, can also be prepared by thismethod. ##STR15##

The electrophilic moiety ("R¹ " in the formula) can also be prepared bymethods known to the art. Such methods may be found in Acc. Chem. Res.17, 202-209 (1984). A process that can prepare compounds of formula (I)where R¹ could be an isothiocyanato moiety (R² or R⁴ present areisothiocyanato when L=formula A) involves reacting the amino chelate (R²or R⁴ equal amino, present when L=formula A) with thiophosgene and isdisclosed in U.S. application Ser. No. 289,172, by M. J. Fazio, et al.,filed Dec. 23, 1988, the disclosure of which is hereby incorporated byreference.

Radionuclides can be produced in several ways. In a nuclear reactor anuclide is bombarded with neutrons to obtain a radionuclide, e.g.

    Sm-152+neutron→Sm-153+gamma

Another method of obtaining radionuclides is to bombard nuclides withparticles in a linear accelerator or a cyclotron. Yet another way is toisolate the radionuclide from a mixture of fission products. The methodof obtaining the nuclides employed in the present invention is notcritical thereto.

The conjugates of the present invention can be prepared by first formingthe complex and then binding the antibody or antibody fragment. Thus theprocess involves preparing or obtaining the ligand, forming the complexwith the metal and then adding the antibody. Alternatively, a processfor making labeled antibody conjugates can involve first conjugation ofthe BFC to the antibody and its subsequent chelation to yield theradionuclide-BFC labeled Ab. Any suitable process that results in theformation of the conjugates of this invention is within the scope ofthis invention.

In the following examples, the following terms and conditions were usedunless otherwise specified.

General Experimental

Mass spectra were obtained on either a Finnigan TSQ mass spectrometer(Q¹ MS mode) or a VG ZAB-MS high resolution mass spectrometer (fast atombombardment with xenon, using 3:1 dithiothreitol:dithioerythritol).

¹ H and ¹³ C NMR spectra were obtained using a Varian VXR-300, BrukerAPC 300, IBM/Bruker NR-80 or a Jeol FX400 spectrometer. All spectra wereobtained at 30° C. unless otherwise noted. ¹ H NMR was done at 300 MHz,80 MHz or 400 MHz, respectively to the equipment listed above; ¹³ C NMRwas done at 75 MHz, 20 MHz or 100 MHz, respectively to the equipmentlisted above. The values for the NMR are δ versus TMS(tetramethylsilane) or when D₂ O was the solvent versus DSS(2,2-dimethyl-2-silapentane-5-sulfonic acid, sodium salt).

Infrared spectra (IR) were recorded on a Nicolet 5SX FT/IR instrument.

For the chromatography procedures, most solvents were Fisher HPLC gradematerials. Ammonium acetate was purchased from Aldrich. Water waspurified using a Barnstead NANOpure™ water filtration system.Preparative chromatography of organic compounds was performed either bynormal gravity chromatography using standard techniques or by flashchromatography as described by C. W. Still et al., J. Org. Chem. 43,2923-24 (1978). The following solvent systems were used:

    ______________________________________    Solvent System  Components    ______________________________________    1               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    2:2:1                    V:V:V    2               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    12:4:1                    V:V:V    3               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    16:4:1                    V:V:V    4               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    4:2:1                    V:V:V    5               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    3:2:1                    V:V:V    6               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    7:3:1                    V:V:V    7               saline (0.85% of NaCl in                    distilled water):conc. NH.sub.4 OH                    4:1                    V:V    8               CHCl.sub.3 :CH.sub.3 OH:conc. NH.sub.4 OH                    4:4:1                    V:V:V    ______________________________________

R_(f) values are reported using these solvent systems and commerciallyavailable, normal phase, silica TLC plates [GHLF 250 micron, AnaltechInc. or Merck Kiesel gel 60F₂₅₄ ]. Preparative column chromatography wasdone using Merck grade 60, 60 Å silica gel.

All percentages are by weight unless stated otherwise.

Some solids were dried using a rotary evaporator (Buchi 461) and/or avacuum oven at a temperature of about 55°-60° C. for several hours. Inaddition, a Virtis model 10-010 automatic freezer dryer or Speed Vac™concentrator was used for solvent removal.

Samarium-153 and lutetium-177 were produced by the Research Reactor,University of Missouri (Columbia, Mo.). Yttrium-90 was purchased fromOak Ridge National Laboratory.

1-(4-Isothiocyanatobenzyl)diethylenetriaminepentaacetic acid(SCN-Bz-DTPA) was prepared by a modification to the procedure of M. W.Brechbiel, et al., Inorg. Chem. 25, 2772-2781 (1986), and purified togive a single species on anion exchange HPLC (Q-Sepharose™). Some of thechemicals used were obtained from the sources indicated:N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), free acidand sodium salt were purchased from Behring Diagnostics (La Jolla,Calif.); sodium citrate (certified) was from Fisher Scientific;thiophosgene was from Aldrich Chemicals; ammonium acetate, sodiumacetate, ethylenediaminetetraacetic acid (EDTA) and phosphate bufferedsaline (PBS) were from Sigma Diagnostics.

HPLC columns used were: Hand-packed Q-Sepharose™ (Pharmacia) either 1.5cm×25 cm or 2.5 cm×25 cm; Zorbax™ BIO Series GF-250 (9.4 mm×25 cm) fromDu Pont Instruments; Vydac™ (Trademark of the Separations Group,Hesperia, Calif.) protein C-4 (4.6 mm×25 cm) from the Separation Group(Hesperia, Calif.); and Mono-Q™ and SP-Sephadex™ (Tradename of PharmaciaBiotechnology Products) from Pharmacia. Sep-Pak™ (Tradename of WatersAssociates) C-18 cartridge was purchased from Waters Associates(Milford, Mass.), Sephadex™ G-25 disposable columns (2.2 ml) from IsolabInc. (Akron, Ohio), and Centricon™-30 (Tradename of Amicon Division, W.R. Grace & Co., Danvers, Mass.) microconcentrators from Amicon.

Five HPLC systems were used for analyses and sample separations:

System I consisted of LKB 2150 pump, and 2152 controller, a UVdetector--LKB 2238 UV Cord, a Berthold LB 506 A HPLC RadioactivityMonitor (of the International Berthold Group) and a Gilson Fractioncollector 201-202 (Gilson International, Middleton, Wis.);

System II was equipped with an auto-sampler, WISP 710 B, two pumps(model 510) and an automated gradient controller (of Waters Associates),a Perkin-Elmer LC-75 spectrophotometric detector, a Beckman model 170Radioisotope Detector, and a fraction collector (Frac-100 of Pharmacia);

System III consisted of two Waters M-6000A pumps with a Waters 660solvent Programmer, a Waters Model 481 Variable Wavelength Detector, anda Waters Data Module, also an ISCO fraction collector was usedpreparatively;

System IV was Dionex BioLC™ System equipped with a variable wavelengthUV detector; and

System V was a Waters Model 590 Pump with an ISCO Model 2360 gradientprogrammer and a Dionex variable wavelength detector.

For centrifugation and concentration, a Sorvall RT 6000B (refrigeratedcentrifuge of Du Pont) was used. A Speed Vac concentrator (SavantInstruments Inc., Hicksville, N.Y.) was employed for removal of volatilesolvents. Radioactivity measurements were done on a Capintec™Radioisotope Calibrator (Trademark of Capintec Inc.) CRC-12, or byliquid scintillation on Beckman LS 9800 (Beckman Instruments, Inc.,Irvine, Calif.), or on a Canberra 35⁺ multichannel analyzer interfacedwith a 3 in. thallium drifted sodium iodide well crystal.

In the examples concerning formation of complex, the percent complexdetermination was performed by the following general method whereindicated.

Percent Complex Determination by Cation Exchange

A 10 ml plastic column was filled with 1 to 2 ml of SP-Sephadex™ C-25resin which was swelled in water. The excess water was removed byapplying pressure to the top of the column. The test solution (15 μl)was added to the top of the resin, then 2 ml of 4:1 (V:V) of isotonicsaline: conc. NH₄ OH was used as an eluent. The eluent was collected ina counting tube. An additional 2 ml of eluent was used. After collectionof eluent into a second counting tube, air pressure was applied to thetop of the resin to bring it to dryness. The dried resin was transferredto a third counting tube. The activity of each counting tube wasmeasured with a NaI well counter coupled to a Canberra multichannelanalyzer. The amount of the metal as a complex was given as thepercentage of the total activity that was found in the eluent. Theamount of metal in the free uncomplexed state was given as thepercentage of the total metal that remains in the resin.

Removal of Uncomplexed Metal by Ion Exchange

A 10 ml plastic column was filled with 0.5 ml of SP-Sephadex™ C-25 resinswelled in water. The excess water was removed by centrifuging thecolumn at 3,000 rpm for 4 min. A volume of 200-500 μl of the complexsolution was placed at the top of the resin and the tube centrifugedagain for 4 minutes at 3,000 rpm. The uncomplexed metal remained in thecolumn and the complexed metal eluted with the solvent.

Yttrium Complex Preparation:

Complexes were made by preparing a 3×10⁻⁴ M yttrium solution in water(YCl₃ 6H₂ O, 303.26 g/mole; or Y(OAc)₃, 12.1% H₂ O). Radioactive Y³⁺solution (Oakridge National Laboratories) was added to give the desiredlevel of radioactivity. Ten μl of ligand solution (at 0.03M) was addedto 990 μl of the Y³⁺ solution, giving a 1:1 molar ratio of ligand:metal.Ten times the amount of ligand solution was used for a 10:1 ligand tometal ratio. The pH was adjusted to 7.4 using microliter quantities ofHCl or NaOH. The solution was then tested for the amount of complexedyttrium using the cation exchange method given above.

Samarium Complex Preparation:

Samarium complexes were formed as described above for yttrium complexesexcept that 3×10⁻⁴ M samarium was prepared by dissolution of Sm₂ O₃(348.7 g/mole) in 0.1M HCl. Radioactive Sm-153 was obtained as a about3×10⁻⁴ M solution in 0.1M HCl from the University of Missouri ResearchReactor, Columbia, Mo.

The following definitions are provided for some terms that are usedthroughout this text.

Glossary:

Conc. means concentrated;

⁻ OAc means the acetate moiety, ⁻ OCOCH₃ ;

TLC means thin layer chromotography;

DI H₂ O means deionized NANOpure™ water;

NANOpure™ water is pure water obtained from a Barnstead NANOpure™ waterfiltration system;

Ambient temperature means room temperature or about 20° to about 25° C.;

Overnight means from about 9 to 18 hours;

LC means liquid chromatography;

NBS means N-bromosuccinimide;

MES means 2-(N-morpholino)ethanesulfonic acid;

HPLC means high performance liquid chromatography;

PBS means phosphate buffered saline from Sigma, containing 120 mM NaCl,2.7 mM KCl and 10 mM phosphate buffer, pH 7.4;

SP-Sephadex™ C-25 resin is a cation exchange resin having sulfonic acidfunctionality, sold by Pharmacia, Inc.;

rpm=revolutions per minute;

pD means pH where the hydrogen is deuterated;

DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

HEPES=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;

BFC=bifunctional chelant;

Cyclen=1,4,7,10-tetraazacylododecane;

PA-DOTA=α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid;

PA-DOTMA=α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid;

BA-DOTA=α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid;

OMe-BA-DOTA=α-(5-amino-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid;

EA-DO3A=1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid;

DTPA=diethylenetriaminepentaacetic acid;

SCN-Bz-DTPA=1-(4-isothiocyanatobenzyl)diethylenetriaminepentaaceticacid;

EDTA=ethylenediaminetetraacetic acid;

chelant is equivalent to ligand;

complex is equivalent to chelate;

conjugate means a chelant or complex covalently attached to an antibodyor antibody fragment; and

antibodies mean CC-49, CC-83 and B72.3 and their fragments such as Faband F(ab')₂. Other possible antibodies are given hereinbefore. Thehybridoma cell line B72.3 is deposited in the American Type CultureCollection, having the accession number ATCC HB 8108, and the othernamed murine monoclonal antibodies bind to epitopes of TAG-72, a tumorassociated antigen.

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of the useof the invention.

Preparation of Starting Materials

EXAMPLE A

Preparation of d,l-2-bromo-4-(4-nitrophenyl)butanoic acid, methyl ester.

To a solution of 5 ml of carbon tetrachloride and 15 ml (0.2 mole) ofthionyl chloride was added 10.46 g (0.05 mole) of4-(4-nitrophenyl)butanoic acid under a nitrogen atmosphere. The solutionwas brought to reflux for 1 hour with initial liberation of hydrogenchloride gas and sulphur dioxide gas. To the warm solution was added11.0 g (0.06 mole) of N-bromosuccinimide in 25 ml of carbontetrachloride and three drops of 48 percent aqueous hydrogen bromidecatalyst. Bromine gas liberation was noted. The dark red solution wasrefluxed for 35 minutes. The solution was cooled and poured into 100 mlof methanol with stirring. TLC analysis (60:40 ethylacetate:hexane v:v)indicated a new product (R_(f) =0.69, silica plates). The excess solventwas removed by rotary evaporation and the dark red oil was filteredthrough a flash silica gel pad (1 in.×5 in.) using methylene chloride asthe eluent. Evaporation of the solvent gave a clear oil, yield 15.43 g,which was a 85:15 mixture of the titled product: methyl ester of thestarting butanoic acid derivative. The titled product was characterizedby:

¹ H NMR (CDCl₃) 8.16(d), 7.38(d), 4.20(dd), 3.79(s), 2.88 (m), 2.38(m);

¹³ C NMR (CDCl₃, 75 MHz) 169.6, 147.5, 129.3, 123.7, 53.0, 44.4, 35.5,33.0.

EXAMPLE B

Preparation ofα-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-acetic acid,1-methyl ester.

To a stirred solution of 1.72 g (10.0 mmole) of1,4,7,10-tetraazacyclododecane in 17 ml of pentene stabilized chloroformwas added 2.07 g (5.82 mmole) of d,l-2-bromo-4-(4-nitrophenyl)butanoicacid, methyl ester (prepared by the procedure of Example A) over fiveminutes under a nitrogen atmosphere with stirring. The reaction mixturewas stirred for 48 hours at about 25° C. TLC (using Solvent System 2, onAnaltech silica gel plates) indicated the formation of the title product(R_(f) =0.73). The yellow chloroform solution was applied to a 1 inch×16inch flash silica gel column (pre-eluted with 5 percent methanolicchloroform), eluted with 250 ml of 5 percent methanolic chloroform,followed by elution with Solvent System 2. Fractions containing puretitle product were combined and evaporated to provide 2.15 g (5.46mmole, 94 percent) of the title product as a light yellow glass.Trituration of a chloroform solution of this glass with diethyl etherresulted in precipitation of a white powder (MP=156°-59° C.) ofanalytical purity and characterized by:

¹ H NMR (CDCl₃) 8.14(d), 7.39(d), 3.71(s), 3.39(dd), 2.5-3.0(m),2.08(m), 2.01(m);

¹³ C NMR (CDCl₃, 75 MHz) 172.7, 149.3, 146.4, 129.2, 123.6, 62.3, 51.2,48.9, 47.2, 45.8, 45,4, 32.8, 30.9.

EXAMPLE C

Preparation of 2-bromo-2-(4-nitrophenyl)ethanoic acid, methyl ester.

A mixture of p-nitrophenylacetic acid (25.0 g, 0.14 mole) and thionylchloride (15.1 ml, 0.21 mole) in dry benzene (100 ml) was stirred atreflux under a N₂ pad for three hours. The mixture was then evaporatedto dryness in vacuo. The residue was dissolved in carbon tetrachlorideand stirred at reflux under a nitrogen pad. Bromine (7.2 ml, 0.14 mole)was added in small portions over a period of three days to the refluxingmixture. The reaction mixture was allowed to cool and the acid chloridewas quenched with methanol (50 ml, added slowly). The resultingbromoester (27 g, 71 percent) was recovered as a yellow oil bydistillation at reduced pressure (BP=168° C. at 1.1 mm) through asix-inch column of glass helices. The structure of the title product wasconfirmed by:

¹ H NMR (CDCl₃) 8.25(d), 7.81(d), 5.51(s), 3.86(s).

EXAMPLE D

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1-aceticacid, 1-methyl ester.

To 529 mg (2.068 mmole) of 2-bromo-2-(4-nitrophenyl)ethanoic acid,methyl ester (prepared by the procedure of Example C) in 20 ml ofacetonitrile was added all at once a solution of 354 mg (2.068 mmole) of1,4,7,10-tetraazacyclododecane in 20 ml of acetonitrile containing justenough methanol to effect complete solution. The resulting pink solutionwas stirred for 1.5 hours then concentrated in vacuo at 35° C. to a lowvolume. This crude monoestertetraamine was purified by silica gelchromatography eluting with 20 percent (wt/v) NH₄ 0Ac/CH₃ OH. Thepurified monoestertetraamine thus isolated as the triacetate salt wasthen converted to the free amine. Thus, 270 mg in 3 ml of water wastreated at 0° C. with aqueous K₂ CO₃ (10 percent wt/wt) to a pH of 11.The basic solution was then extracted with 10 ml of chloroform, fivetimes, and the chloroform layers combined, dried over sodium sulfate andconcentrated to give 160 mg (42 percent yield) of the desiredmonoestertetraamine, characterized by NMR and fast atom bombardment massspectrometry ([M+H]⁺ =366) and by:

¹ H NMR (CDCl₃) 8.12(d), 7.44(d), 4.75(s), 3.76(s), 2.67-2.44(m); ¹³ CNMR (CDCl₃) 171.5, 147.6, 144.0, 130.0, 124.0, 67.0 49.8, 47.9, 46.9,46.0.

EXAMPLE E

Preparation ofN-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane.

To a stirred chloroform solution (20 ml) containing 2.9 g of1,4,7,10-tetraazacyclododecane (16.8 mmole) was added a chloroformsolution (20 ml) containing 2.1 g of 2-methoxy-5-nitrobenzyl bromide(8.5 mmole) in one portion. After stirring at room temperature for threehours the reaction mixture was filtered and the filtrate concentrated(in vacuo) to give a residue which was chromatographed (silica, SolventSystem 3). The monoalkylated product was isolated in 79 percent yield(MP=154°-156° C.), and characterized by:

¹³ C NMR (CDCl₃) 162.47, 140.63, 127.92, 125.49, 124.53, 109.91, 55.88,53.25, 50.90, 47.18, 45.45, 45.29.

EXAMPLE F

Preparation ofN-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane.

To a 1,4-dioxane solution (20 ml) of 1,4,7,10-tetraazacyclododecane (2.3g, 14 mmole) was added a dioxane solution (15 ml) of2-hydroxy-5-nitrobenzyl bromide (1.2 g, 7 mmole) in one portion withconstant stirring. After several minutes K₂ CO₃ (1 g) was added andstirring continued for 1 hour at room temperature. The reaction mixturewas then filtered and the filtrate concentrated in vacuo to give ayellow semi-solid which was chromatographed (column) on silica geleluting with Solvent System 5. The desired mono-alkylated product wasisolated from the last 1/3 of bright yellow eluent which also containedunreacted amine. After concentration, the yellow oil was triturated withCDCl₃ (100 ml) and filtered to remove CDCl₃ -soluble amine startingmaterial. The final product was then isolated in pure form as a yellowsolid (1.2 g, 55 percent); (MP=142°-145° C.) and further characterizedby:

¹³ C NMR (D₂ O) 25.04, 25.28, 26.83, 31.80, 36.54, 101.88, 107.22,110.92, 111.80, 115.26, 159.99.

EXAMPLE G

Preparation of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane.

To a stirred solution of 3.50 g (20.3 mmole) of1,4,7,10-tetraazacyclododecane in 50 ml of pentene stabilized chloroformwas added dropwise over a five minute period with vigorous stirringunder a nitrogen atmosphere 4.00 g (17.4 mmole) of1-(2-bromoethyl)-4-nitrobenzene.

Stirring was continued for about 18 hours at room temperature (about 25°C.) whereupon crystals of amine hydrobromide precipitated from solution.The entire reaction mixture was applied to a flash silica gel column (1in×18 in) which had been pre-eluted with 5 percent methanol inchloroform; 200 ml of this solution was applied as an eluent, followedby elution with Solvent System 3. From the desired product was separated1.45 g (9.7 mmole) of p-nitrostyrene (R_(f) =0.98; Solvent System 1).The desired monofunctional title product was isolated as anorange-yellow oil (2.27 g, 7.06 mmole, 40.6 percent) which solidifiedupon standing (R_(f) =0.73, Solvent System 1).

A sample of the titled product was recrystallized from CHCl₃/cyclohexane and showed the following characteristics: MP=146.5°-148.5°C.;

¹ H NMR (CDCl₃) 8.135(d, m), 7.395(d, m), 2.91(t), 2.77(t), 2.72(t),2.50(t), 2.60(s);

¹³ C NMR (CDCl₃, 75 MHz) 148.5, 146.7, 129.6, 123.4, 55.5, 51.4, 46.9,45.9, 45.1, 33.7.

EXAMPLE H

Preparation of 1-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecane.

In 50 ml of chloroform was added 3.5 g (20.3 mmole) of1,4,7,10-tetraazacyclododecane and 1.7 g (7.87 mmole) p-nitrobenzylbromide and the mixture stirred under nitrogen for 24 hours at 25° C.The chloroform slurry of hydrobromide salt was then applied to a 1in.×17 in. column of flash silica gel (Solvent System 3). There wasobtained 2.13 g (6.93 mmole) of the title product as a pale yellow solidof analytical purity in 88 percent yield (R_(f) =0.58, Solvent System3), MP=128°-129° C., and further characterized by:

¹ H NMR (CDCl₃) 8.19 (d), 7.49 (d), 3.69 (s), 2.82 (t), 2.70 (t), 2.59(m);

¹³ C NMR (CDCl₃, 75 MHz) 147.2, 128.4, 123.8, 58.8, 51.7, 47.1, 46.3,45.1.

(No Example I; to avoid confusion with later biology example numers).

EXAMPLE J

Preparation of 1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane.

In 10 ml of methanol was dissolved 690 mg (2.25 mmole) of1-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecane (prepared by theprocedure of Example H). To the solution was added 350 mg of 10 percentpalladium on carbon catalyst. Excess hydrogen was purged through thesolution at 25° C. Within 45 minutes TLC indicated that the titleproduct was prepared (R_(f) =0.33, Solvent System 3). Chromatographicpurification of the formed titled product was conducted on 1 in.×16 in.flash silica column (Solvent System 3) and provided 480 mg (77 percent)of the title product as a pale yellow, clear oil.

The free base title product (103 mg, 0.37 mmole) was converted to thehydrochloride salt by bubbling anhydrous hydrogen chloride throughethanol solutions of the free base. The resulting tetrahydrochloridesalt of the title product, after washing with cold ethanol and drying invacuo, was obtained in a yield of 147 mg (0.347 mmole), MP=255°-260° C.(dec), and was further characterized by:

¹ H D₂ O, pD=1.9) 7.56 (d), 7.47 (d), 3.95 (s), 3.28 (t), 3.23 (t), 3.09(t), 2.96 (t);

¹³ C NMR D₂ O, pD=1.9, 75 MHz) 138.3, 134.2, 132.2, 126.0, 58.5, 50.3,46.7, 44.6, 44.3.

EXAMPLE K

Preparation of 2-methoxy-5-nitrobenzylnitrile.

To a solution of sodium cyanide (6 g, 121.9 mmole) in water (5.3 ml) at70° C. was added in small portions with stirring a hot solution of2-methoxy-5-nitrobenzylbromide (25 g., 101.6 mmole) in ethanol (15 ml).The reaction mixture was then refluxed for 1.5 hours, cooled, andfiltered. The filter cake was washed with acetonitrile and the filtratewas evaporated in vacuo to yield 2-methoxy-5-nitrobenzylnitrile (19.5g., 100 percent) as a tan solid. The product was characterized by:

¹³ C NMR (CDCl₃) 161.4, 141.0, 125.7, 124.6, 119.8, 116.5, 110.2, 56.3,18.8.

EXAMPLE L

Preparation of 2-methoxy-5-nitrophenylacetic acid.

A slurry of 2-methoxy-5-nitrobenzylnitrile (19.5 g) (prepared by theprocedure of Example K) in conc. HCl (20 ml) was refluxed for threehours. After cooling, the crude product was recovered by filtration andwashed with water. The solid was taken up in hot aqueous sodiumhydroxide and filtered while hot. The orange solution was cooled andacidified with HCl. The precipitate was filtered, washed with water, anddried to yield 2-methoxy-5-nitrophenylacetic acid as a white solid (15g, 70 percent). The product was characterized by:

¹³ C NMR (CDCl₃ -CD₃ OD) 172.8, 162.5, 140.7, 126.2, 124.7, 124.1,109.7, 55.8, 35.0.

EXAMPLE M

Preparation of α-bromo-(2-methoxy-5-nitrophenyl)acetic acid, methylester.

To a slurry of 2-methoxy-5-nitrophenylacetic acid (2,266 g, 10.74 mmole)(prepared by the procedure of Example L) in dry benzene (50 ml) wasadded thionyl chloride (4.0 ml, 54.8 mmole). A drying tube and condenserwere placed on the flask and the mixture was refluxed for 2 hours. Theresulting yellow solution was concentrated in vacuo to a small volumeand taken up in dry carbon tetrachloride. Bromine (0.6 ml, 11.6 mmole)was added and the solution was refluxed for two days with the exclusionof atmospheric moisture. The reaction mixture was concentrated to asmall volume and methanol (50 ml) was added. After evaporation of excessmethanol in vacuo the resulting oil was purified chromatographically(silica gel, methylene chloride-hexane 4:1). The title product (2.399g., 74 percent) was obtained as a yellow oil. The product wascharacterized by:

¹ H NMR (CDCl₃) 8.45(dd), 8.15(dd), 6.95 (d), 5.75(s), 3.99(s), 3.78(s).

EXAMPLE N

Preparation ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane, 1-aceticacid, methyl ester.

A solution of α-bromo (2-methoxy-5-nitrophenyl)acetic (2.399 g, 7.89mmole) (prepared by the procedure of Example M) in chloroform (10 ml)was added to a stirred solution of 1,4,7,10-tetraazacyclododecane (2.72g, 15.79 mmole) in chloroform (50 ml). The mixture was stirred at roomtemperature for 2 hours. The cloudy mixture was then concentrated to asmall volumn in vacuo at room temperature. The crude product waspurified by chromatography (silica gel, Solvent System 3). The titlecompound was recovered as a yellow solid (2.60 g, 83 percent) andcharacterized by:

¹³ C NMR (CDCl₃) 170.7, 161.6, 139.9, 125.0, 124.5, 124.2, 110.2, 59.5,55.5, 50.5, 48.0, 47.3, 45.6, 44.3, 43.5.

EXAMPLE O

Preparation of d,l-2-bromo-4-(4-nitrophenyl)butanoic acid isopropylester.

4-(4-Nitrophenyl)butanoic acid (21.0 g, 0.10 mole) was added to asolution of carbon tetrachloride (10 ml) and thionyl chloride (30 ml,0.4 mole) under a nitrogen atmosphere using the procedure of Harpp etal., J. Org. Chem 40, 3420-27 (1975). The solution was brought to refluxfor 1 hour with initial rapid liberation of hydrogen chloride and sulfurdioxide. At this point, N-bromosuccinimide (22.0 g, 0.12 mole) was addedas a solution in carbon tetrachloride (50 ml) and 8 drops of 48% aqueoushydrogen bromide catalyst was added to the warm solution whereuponbromine liberation was noted. The dark red solution was refluxed for anadditional 35 minutes. The solution was cooled and poured intoisopropanol (400 ml) with stirring. TLC analysis (methylene chloride,silica gel plates) revealed a new product (R_(f) =0.73). The excesssolvent was removed and the dark red oil was filtered through a flashsilica gel pad (3 in.×6 in.) using methylene chloride as an eluent. Thesolvent was removed in vacuo and the light yellow oil was applied to aflash silica gel column (3 in.×18 in.) and eluted with methylenechloride to afford 25.0 g (0.076 mole) of the titled bromoester productas a clear oil in 75% yield as a isopropanol solvate which containedless than 5% of unbrominated ester and characterized by:

¹ H NMR (CDCl₃) 8.16(d, 2H), 7.38(d, 2H), 5.05(septet, 1H), 4.14(dd,1H), 2.88(m, 4H), 2.39(m, 4H), 1.29(d, 6H);

¹³ C NMR (CDCl₃) 168.7, 147.7, 129.3, 123.8, 69.9, 45.1, 35.6, 33.0,21.5, 21.2.

EXAMPLE P

Preparation ofα-[3-(4-nitrophenyl)propyl]-1,4,7,10-tetraazacyclododecane-1-aceticacid, 1-methyl ester.

To a stirred solution of 3.137 g (18.2 mmoles) of cyclen free base in 30ml of pentene stabilized chloroform was added 4.81 g (14.6 mmolescorrected) of d,l-2-bromo-4-(4-nitrophenyl)-butanoic acid isopropylester (prepared by the procedure of Example O) over a 5 minute periodunder a nitrogen atmosphere with stirring. The reaction solution wasstirred for 24 hours at room temperature. TLC analysis (Solvent System2) revealed conversion to the titled monoalkylation product (R_(f) =0.78detection withninhydrin, iodine, and UV activity). The yellow chloroformsolution was applied to a 1 in.×17 in. flash silica gel column which hadbeen pre-eluted with 5% methanolic chloroform. Elution with 300 ml ofthis solvent system was followed by elution with Solvent System 2 andprovided fractions containing pure titled product which were combinedand evaporated affording 4.85 g (5.46 mmoles) of the titled product freebase as a light oil in 79% yield and further characterized by:

¹ H NMR (CDCl₃) 8.15(d, 2H), 7.40(d, 2H), 5.07(p, 1H), 3.35(dd, 1H),2.65-3.0(m, 13H), 2.5-2.64(m, 4H), 2.14(m, 1H), 2.00(m, 1H), 1.28(dd,6H);

¹³ C NMR (CDCl₃) 171.6, 149.5, 146.5, 129.2, 123.6, 68.1, 62.7, 49.2,47.5, 45.9, 45.7, 32.9, 31.0, 22.1, 22.0;

IR (CDCl₃) cm⁻¹ 3231(N--H), 2978, 2933, 1721(ester carbonyl), 1.601,1512, 1458, 1345, 1107;

Fast atom bombardment mass spectrum, m/e 422 (M+H)]+, 408, 392.

Preparation of Final Products--Ligands

EXAMPLE 1

Preparation ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, monomethyl, triethyl ester.

In 46 ml of acetonitrile containing 4.6 g (33.3 mmole) of freshlypowdered anhydrous potassium carbonate was dissolved 700 mg (1.91 mmole)of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid,1-methyl ester (prepared by the procedure of Example D). To thissuspension was added all at once 1.022 g (6.12 mmole) of ethylbromoacetate. After four hours of stirring at room temperature, thesuspension was vacuum filtered, the filter cake washed with 15 ml ofacetonitrile, two times, and the filtrate evaporated in vacuo at 38° C.to give the crude titled tetraester as a purple solid. The tetraesterwas then purified by silica gel chromatography eluting with 5 percent C₂H₅ OH/CHCl₃ then Solvent System 3, to give 620 mg (0.988 mmole, 52percent) of the desired product. This product was characterized by fastatom bombardment mass spectrometry ([M+H]⁺ =624) and futhercharacterized by:

¹ H NMR (CDCl₃) 8.24(d), 7.45(d), 4.94(s), 4.35-4.10(m), 3.79(s),3.75-1.84(m), 1.34-1.22(m);

¹³ C NMR (CDCl₃) 174.3, 173.8, 173.6, 171.5, 147.6, 138.9, 131.5, 123.4,64.8, 61.5, 61.4, 60.2, 55.4, 55.0, 53.1, 52.9, 52.7, 52.4, 51.9, 51.7,48.8, 48.3, 44.9, 19.1, 14.1.

EXAMPLE 2

Preparation ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.

In 30 ml of 6N HCl was dissolved 300 mg (0.478 mmole) ofa-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, monomethyl, triethyl ester (prepared by the procedure ofExample 1) and the mixture was heated at reflux overnight. At the end ofthis reflux period, the solution was concentrated in vacuo at 70° C. togive a yellow solid. This solid was dissolved in 3 ml of water,filtered, and the filtrate evaporated to give 253 mg (0.378 mmole, 79percent) of the crude product. This product was purified by preparativeTLC [silica gel, developed in 20 percent NH₄ 0Ac/CH₃ OH (wt/v)] andcharacterized by fast atom bombardment mass spectrometry ([M+H]⁺ =526)and futher characterized by:

¹ H NMR (D₂ O) 8.21(d), 7.42(d), 4.83(s), 3.83-2.19(m), 1.92(s);

¹³ C NMR (D₂ O) 75.0, 173.6, 171.8, 167.0, 166.3, 146.9, 138.3, 130.1,123.0, 62.2, 54.0, 53.0, 52.2, 50.4, 97.3, 46.8, 44.8, 42.8, 20.0.

EXAMPLE 3

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid; (BA-DOTA).

Method A:

Catalytic hydrogenation of the nitro group of the tetraacid (prepared bythe procedure of Example 2) was performed using Adams catalyst (PtO₂)and hydrogen in essentially quantitative yield to give the titledproduct. This product was characterized by fast atom bombardment ([M+H]⁺=496) and anion exchange HPLC (on Q-Sepharose™) and futher characterizedby:

¹ H NMR (D₂ O) 8.12(d), 7.86(d), 5.66(s), 4.73-4.57(m), 4.27-3.78 (m),3.39-2.91(m);

¹³ C NMR (D₂ O) 176.54, 176.52, 176.46, 141.1, 128.9, 120.9, 112.5,65.0, 55.9, 55.7, 53.6, 49.7, 49.5, 49.3, 45.7, 45.4, 44.9, 44.6, 41.4.

Method B:

Another method to synthesize this compound was to convert the monoestertetraamine compound from Example D to the monoacid tetraamine compound(6N HCl, reflux overnight), followed by aqueous alkylation usingbromoacetic acid, then reduction of the nitro group to the amine group(PtO₂ catalyst) to give a product identical to that described above.

EXAMPLE 4

Preparation ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

In 60 ml of 6N HCl was dissolved 2.0 g (5.48 mmole) of the monoestertetraamine compound (prepared by the procedure of Example D). Themixture was heated to 95° C. overnight (about 10 hours) after which itwas concentrated in vacuo at 75° C. to give 2.53 g (5.11 mmole, 96percent) of the monoacid tetraamine tetrahydrochloride as a yellowsolid.

The above monoacid tetraamine tetrahydrochloride (0.5 g, 1.0 mmole) wasdissolved in 15 ml of water and adjusted to pH 9 with NaOH. A separatesolution of bromoacetic acid was prepared (0.71 g, 5.13 mmole) and addedto the water solution of the monoacid tetraamine. The pH of the solutionwas again adjusted to 9 and maintained at this pH during the reaction bysmall additions of 1.0N NaOH. While the reaction was stirred, aliquotswere removed at different time points and the progress of the alkylationwas monitored by anion exchange HPLC. When the amount of dialkylationproduct (total of three acetate groups present) had reached a maximum,the whole reaction mixture was lyophilized to give a dark solid. Thiscrude mixture of alkylated products was then purified by silica gelchromatography (eluting with Solvent System 1), followed by preparativeanion exchange chromatography (eluting with a gradient of 0-100 percent1M NH₄ OAc), then by preparative TLC plates (developed in Solvent System4) and finally by silica gel chromatography (eluting with Solvent System4). This extensive purification procedure gave, as a yellow solid, 30 mg(0.06 mmole, 6.0 percent) of the title product. This product wascharacterized by fast atom bombardment mass spectrometry ([M+H]⁺ =468),anion exchange HPLC and futher characterized by:

¹ H NMR (D₂ O) 8.12(bs), 7.35(bs), 4.76(s), 3.57-3.44(m), 2.97-1.83(m),1.80(s);

¹³ C NMR (D₂ O) 181.75, 180.68, 179.00, 175.37, 147.70, 141.22, 133.08,123.53, 68.44, 59.72, 56.00, 52.80, 49.96, 49.29, 47.86, 45.36, 44.26,42.78, 42.25, 23.72.

EXAMPLE 5

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidpentahydrochloride andα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acidpentahydrochloride.

A crude mixture of alkylated products was prepared by the proceduredescribed in Example 4. This crude mixture was chromatographed on asilica gel column eluting with 20 percent NH₄ OAc/CH₃ OH (wt:v). Theappropriate fractions from this column were combined and evaporated todryness. The crude product, having substantial amounts of NH₄ OAc, wasdissolved in 80 ml of NANOpure™ water and lyophilized to yield a lightbrown solid. This solid was dissolved in 20 ml of NANOpure™ water,shaken with PtO₂ catalyst under a H₂ atmosphere until hydrogen uptakestopped. The catalyst was separated by vacuum filtration, and thefiltrate lyophilized to give a yellow solid. The solid was dissolved in3 ml of Solvent System 4 and chromatographed on silica gel using SolventSystem 4. The pooled fractions were then stripped in vacuo andlyophilized to yield 779.6 mg of light yellow solid. ¹³ C NMR and protonNMR suggests that this product is a 50/50 mixture of the two possiblegeometric isomers. The isomeric mixture was contacted with excess HCland lyophilized to yield 548.4 mg (61 percent yield) of the titleproducts. M.P.>270° C. Anion exchange chromatography (HPLC System III)showed one major peak (>94 percent purity); fast atom bombardment massspectrum indicated the geometrical isomers [M+H]⁺ =438.

EXAMPLE 6

Preparation ofα-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester.

To a solution of 1.43 g (3.63 mole) ofα-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-acetic acid,1-methyl ester (prepared by the procedure of Example B) in 40 ml ofargon purged acetonitrile was added 1.71 g (12.34 mmole) of anhydrouspotassium carbonate with vigorous stirring. To the reaction mixture wasadded 1.78 g (11.61 mmole) of methyl bromoacetate and the reactionmixture was stirred at about 25° C. for 48 hours. TLC analysis indicatedformation of a new product (R_(f) =0.62, Solvent System 2). To theslurry was added 6.0 g of flash silica gel and acetonitrile was removedon a rotary evaporator. The resulting material was applied to 1 in.×16in. silica column which had been pre-eluted with 5 percent methanolicchloroform. About 400 ml of the prepared solution was used to eluteseveral nonpolar impurities and unreacted alkylating agent. Then SolventSystem 2 was applied, fractions containing product (R_(f) =0.62) werecollected, combined and evaporated to provide 2.1 g (3.44 mmole, 95percent) of the titled product as a pale yellow glass. ¹ H NMR indicatedthis product to be a 2:1 mixture of conformational or geometric isomers.

¹ H NMR (CDCL₃, 50° C.) 8.137(d), 8.128(d), 7.398(d), 7.385(d), 3.82(s),3.76(s), 3.75(s), 3.74(s), 3.68(s), 1.5-3.52(m);

¹³ C NMR (CDCl₃, 50° C., 75 MHz) 175.8, 174.2, 174.1, 174.0, 149.0,129.5, 129.3, 123.6, 60.1, 55.1, 52.8, 52.7, 52.6, 52.4, 52.2, 52.1,52.0, 51.2, 51.1, 51.0, 49.1, 48.8, 47.5, 45.2, 34.2, 32.6.

EXAMPLE 7

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester.

In 25 ml of methanol containing 400 mg of 10 percent palladium-on-carboncatalyst under a nitrogen atmosphere was dissolved 1.41 g (2.31 mole) ofα-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester (prepared by the procedure of Example6). Excess hydrogen was purged through the solution at one atmospherefor three hours. TLC indicated the formation of a strongly ninhydrinpositive material (R_(f) =0.62, Solvent System 2). The methanoliccatalyst slurry was filtered through celite and evaporation of solventprovided the title product, 1.21 g (2.08 mmole, 91 percent), as a whitesolid glass (as a 2:1 mixture of conformational isomers). The minorconformational or geometric isomer was separated from the major isomerby flash chromatrography using 10 percent methanolic chloroform to yieldthe title product as an off white powder (MP=89°-95° C.) andcharacterized by:

¹ H NMR (CDCl₃, 50° C.) 6.94(d), 6.89(d), 6.69(d), 6.66(d), 3.91(s),3.80(s), 3.78(s), 3.74(s), 3.73(s), 3.72(s), 3.71(s), 1.5-3.5(m);

¹³ C NMR (CDCl₃, 50° C., 75 MHz) 176.1, 174.0, 173.9, 172.2, 170.4,169.6, 144.2, 144.1, 130.6, 130.5, 129.5, 129.1, 115.9, 115.8, 62.6,58.7, 55.1, 54.3, 52.7, 52.5, 52.3, 52.2, 52.1, 52.0, 51.9, 51.8, 51.7,50.2, 50.0, 47.3, 44.7, 32.8, 31.8, 30.0, 25.2;

Fast atom bombardment mass spectrum, m/e 602 [M+Na⁺ ], 618 [M+K⁺ ]⁺,[624 M+2Na⁺ -H⁺ ]⁺.

EXAMPLE 8

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid; (PA-DOTA).

In 50 ml of conc. hydrodrochloric acid was dissolved 1.5 g (2.59 mmole)ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester (as the crude 2:1 mixture prepared bythe procedure of Example 7). The reaction mixture was refluxed at about105° C. for six hours. TLC (Solvent System 1) indicated conversion ofthe ester (R_(f) =0.88) to the title product as the hydrochloride salt(R_(f) =0.43). Excess solvent was removed on a rotary evaporator and theresulting white solid was dried. The title product as the hydrochloridesalt was obtained in a yield of 1.6 g and characterized by:

¹ H NMR D₂ O, pD=1.0., 90° C.) 7.48(d), 7.42(d), 2.8-4.3(m), 2.23(m),2.03(m);

¹³ C NMR D₂ O, pD=1.0, 90° C., 75 MHz) 176.4, 174.9, 171.6, 144.7,132.9, 132.8, 130.4, 125.7, 61.7, 56.8, 56.0, 53.7, 53.1, 51.6, 47.9,34.3, 37.6,

Fast atom bombardment mass spectrum m/e 524 [M+H]⁺, 546 [M+Na⁺ ]⁺, 568[M+2Na⁺ -H⁺ ]⁺.

Using flash chromatography and Solvent System 1, any trace impurities ofthe ester were removed from the title product as the hydrochloride salt.To a 1 in.×23 in. flash silica gel column was applied 1.10 g of thetitle product, as the hydrochloride salt. Fractions containing only thetitle product, as the mixed ammonium potassium salt were combined toprovide 580 mg.

HPLC analysis indicated the material to be greater than 98 percent areapurity at 230 and 254 nm.

Fast atom bombardment mass spectrum, m/e 562 (M+K)⁺, 584 [M+K⁺ +Na⁺ -H⁺], 600 [M+2K⁺ -H⁺ ]⁺.

EXAMPLE 9

Isolation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid, mixed ammonium, potassium salt.

A crude solution containing both the tetra and tri acids (2.05 g)(prepared by the procedure of Example 8) was dissolved in a minimumamount of Solvent System 1 and applied to a 12 in.×3 in. column of flashsilica gel which had been pre-eluted with this solvent. Elution offractions containing the title product (R_(f) =0.63 in Solvent System 1)were collected and afforded 200 mg ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid, mixed ammonium, potassium salt. ¹ H and ¹³ C NMR analysissuggested that this triacid was the symmetrical isomer (1,4,10-triacidpositional isomer):

¹ H NMR D₂ O, pD=0.5 with DCl, T=90° C.) 7.52(d), 7.46(d), 3.60(m),3.54(m), 3.19(m);

¹³ C NMR D₂ O, pD=0.5, T=90° C.) 176.1, 170.2, 140.0, 132.7, 131.1,126.1, 57.6, 57.2, 56.1, 54.9, 53.2, 51.5, 51.2, 31.2.

Fast atom bombardment mass spectrum, m/e 466[M+H⁺ ]⁺, 488[M+Na^(+]) ⁺,504[M+K⁺ ]⁺, 526[M+K⁺ +-H⁺ ]⁺, 542[M+2K-H)]⁺.

EXAMPLE 10

Preparation ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, tetramethyl ester.

Methyl bromoacetate (565 μl, 5.97 mmole) was added to a stirred mixtureof α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane, 1-aceticacid, methyl ester (580 mg, 1.47 mmole) (prepared by the procedure ofExample N) and pulverized potassium carbonate (1.15 g, 8.32 mmole) inacetonitrile (20 ml). The reaction mixture was stirred at roomtemperature for 2 hours. The mixture was then filtered and the filtratewas concentrated to an oil in vacuo. The crude product was purified bychromatography (silica gel, 8 percent methanol in methylene chloride).The title product was obtained as a yellow solid (469 mg, 52 percent),TLC R_(f) =0.4 (silica gel, 10% CH₃ OH in CHCl₃), and furthercharacterized by:

13C NMR (CDCl₃) 173.2, 172.6, 161.2, 139.1, 125.1, 124.6, 120.5, 110.7,57.0, 55.6, 53.5, 52.6, 51.3, 50.8, 46.8, 46.0, 44.0.

EXAMPLE 11

Preparation ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.

A solution ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, tetramethyl ester (prepared by the procedure of Example 10) inconcentrated HCl (5 ml, J. T. Baker ULTREX) was refluxed under anitrogen atmosphere for 5 hours. The solution was then concentrated todryness in vacuo to leave a solid residue. This was purified bychromatography (silica gel, Solvent System 6) to yield the title productas an off-white solid (209 mg). This material was converted to thetetrahydrochloride salt and was characterized by:

¹³ C NMR (D₂ O-DCl, 80° C.) 171.0, 166.9, 161.2, 139.0, 125.5, 119.3,110.7, 56.8, 54.5, 52.7, 51.0, 49.2, 49.0, 46.3, 42.9.

EXAMPLE 12

Preparation ofα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, tetraammonium salt.

To a solution ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (157 mg) (prepared by the procedure of Example 11) in water (20 ml)was added platinum oxide (20 mg). This mixture was hydrogenated (1atmosphere hydrogen) for 1 hour at room temperature. After filtration,the material was further purified by chromatography (silica gel, SolventSystem 5) to yield the title compound (141 mg) as an off-white solid,and characterized by:

¹³ C NMR (D₂ O)-DCl) 175.5, 172.6, 172.3, 159.9, 128.8, 127.5, 124.6,123.8, 115.5, 61.2, 58.1, 57.3, 55.7, 53.4, 51.6, 49.6, 47.4.

EXAMPLE 13

Preparation of1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid,4,7,10-trimethyl ester.

To 4 ml of argon purged acetonitrile with stirring was added 100 mg(0.236 mmole) of 1-(4-aminobenzyl)-1,4,7-10-tetraazacyclododecane,tetrahydrochloride salt (prepared by the procedure of Example J), 260 mg(1.88 mmole) of potassium carbonate, and 108 mg (0.708 mmole) ofmethylbromoacetate. The mixture was stirred for 48 hours at 25° C. Thesalt containing solution was applied to a 1 cm×4 cm flash silica gelcolumn and was eluted with acetonitrile. Fractions containing thedesired product were combined and the solvent removed by rotaryevaporation to provide 65 mg (0.132 mmole, 56 percent) of the titleproduct which was further characterized by:

¹ H NMR (CDCl₃) 7.29 (d), 6.75 (d), 4.62 (s), 4.20 (broad s), 3.70 (s),3.69 (s), 3.64 (s), 3.62 (t), 3.27 (s), 3.06 (t), 2.82 (broad t), 2.79(broad t);

¹³ C NMR (CDCl₃, 75 MHz) 171.4, 171.0, 148.4, 132.8, 117.5, 115.0, 55.9,55.3, 54.7, 53.1, 51.7, 51.4, 50.6, 50.5, 47.4.

EXAMPLE 14

Preparation of1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid,hydrochloride salt.

In 2 ml of 6N hydrochloric acid, 42 mg (0.085 mmole) of1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid,4,7,10-trimethyl ester (prepared by the procedure of Example 13) washeated to 80° C. for 2 hours. TLC indicated several products (R_(f)=0.60 for the desired title product, Solvent System 1). Removal ofsolvent afforded 41 mg of crude title product.

EXAMPLE 15

Preparation of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10,-triaceticacid, 4,7,10-trimethyl ester.

To a solution of 2.24 g (6.97 mmole) of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane (prepared bythe procedure of Example G) in 75 ml of argon purged acetonitrile wasadded with vigorous stirring 3.17 g of anhydrous potassium carbonate. Tothis reaction mixture was added dropwise over a five minute period 3.20g (20.9 mmole) of methylbromoacetate. The reaction mixture was stirredfor 17 hours under argon atmosphere. TLC analysis showed conversion ofthe starting material (R_(f) =0.73, Solvent System 1) to a new product(R_(f) =0.46, Solvent System 1)o To the solution was added 70 ml ofchloroform and the solution with the suspended salt was applied to a 1in.×7 in. flash silica column. The product was eluted using 10 percentmethanol in chloroform to yield 2.95 g of the title product as an amberoil which formed a friable glass upon vacuum drying (78 percent). Thetitle product was characterized by:

¹ H NMR (CDCl₃) 8.17 (m,m), 7.4-7.66 (m), 2.38-3.95 (m).

¹³ C NMR (CDCl₃, 75 MHz) 171.5, 171.2, 146.7, 144.5, 130.3, 123.9, 56.0,55.2, 53.5, 53.4, 52.6, 51.9, 51.8, 50.6, 48.1, 29.5.

EXAMPLE 16

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, 4,7,10-trimethyl ester.

In 50 ml of methanol was dissolved 2.8 g (5.18 mmole) of crude1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, 4,7,10-trimethyl ester (prepared by the procedure of Example 15).To the stirred solution which was purged with nitrogen was added 10percent palladium on carbon catalyst (600 mg). The solution wasmaintained under an atmosphere of nitrogen, and then hydrogen (1 atm,20°-25° C.) was purged through the stirred solution for 2.5 hours. Thesolution was then purged for several minutes with nitrogen and thecatalyst removed by filtration through a short bed of celite. TLCanalysis (10 percent methanol in chloroform) revealed the title product(R_(f) =0.14). The title product was eluted with chloroform from silicagel to yield 2.2 g (4.33 mmole, 83 percent) as a white glass andcharacterized by:

¹ H NMR (CDCl₃) 7.03 (d,m), 6.63 (d), 3.4-3.6 (m), 2.7-3.2 (m);

¹³ C NMR (CDCl₃, 75 MHz) 171.4, 171.2, 145.6, 129.7, 125.6, 115.5, 55.9,54.4, 54.3, 53.0, 52.8, 51.8, 51.6, 50.3, 47.8, 28.6;

Fast atom bombardment mass spectrum, m/e 508 [M+H⁺ ]⁺.

EXAMPLE 17

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, hydrochloride salt; (EA-DO3A).

In 30 ml of conc. HCl was dissolved 850 mg (1.69 mmole) of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, 4,7,10-trimethyl ester (prepared by the procedure of Example 16).The solution was stirred at 70°-80° C. for 2.5 hours and the solutionallowed to cool with stirring to 25° C. and the stirring continued forabout 18 hours. Solvent was removed on a rotary evaporator at reducedpressure (10 mm, 75° C.). Solvate and excess hydrogen chloride wereremoved from the resulting clear oil by vacuum drying (10⁻¹ mm, 45° C.).The title product was provided as a white solid, in a yield of 890 mg,(R_(f) =0.46, Solvent System 2) and further characterized by:

¹ H NMR D₂ O, pD=1.9, T=90° C.) 7.50(d), 7.41(d), 4.26(s), 3.43-3.68(m),3.0-3.3(m);

13C NMR D₂ O, pD=1.9, T=90° C.) 176.7, 170.9 139.8, 133.0, 131.2, 125.9,57.5, 57.1, 55.4, 54.4, 52.7, 51.0, 50.6, 31.3;

Fast atom bombardment mass spectrum, m/e 466 (M+H⁺), 488 [M+Na⁺ ], 510[M+2Na⁺ -H⁺ ]⁺.

HPLC analysis indicated greater than 92 percent area purity (254 mm)using a 10 cm Partisil-5 OD53 RAC II reverse phase column. The eluentwas 10 percent acetonitrile in 0.05M pH=7.0 potassium phosphatesolution.

EXAMPLE 18

Preparation of1-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid; (SCN-EA-DO3A).

1-[2-(4-Aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, hydrochloride salt (prepared by the procedure of Example 17, 106mg, 0.21 mmole) was dissolved in 2 ml of water under a nitrogenatmosphere with stirring. Sodium bicarbonate (105.6 mg, 1.26 mmole) wasslowly added to prevent frothing from carbon dioxide evolution(resulting pH=8.0). Thiophosgene (16.8 μl, 0.22 mmole) was added andafter one hour of vigorous stirring, TLC analysis (20 percent water inacetonitrile) indicated conversion of starting material (R_(f) =0.12) totitle product (R_(f) =0.26). The solvent was removed from the crudeproduct by rotary evaporation to afford 130 mg of title product whichcontained sodium chloride. Infrared analysis of this material (KBrpellet) confirmed the presence of the isothiocyanate moiety (SCN=2150cm⁻¹ ).

EXAMPLE 19

Preparation of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, triethyl ester.

To 5 ml of a stirred acetonitrile solution containing 395 mg (1.12mmole) of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane(prepared by the procedure of Example G) and 1.5 g (11 mmole) of K₂ CO₃was added 496 μl (4.5 mmole) of ethyl bromoacetate in one portion. Afterheating at 68° C. under a N₂ atmosphere for one hour, the resultingsuspension was filtered. The filter cake was washed with CH₃ CN (2×10ml). The filtrate was then concentrated to give the crude product as aviscous oil, triturated with 30 ml of diethyl ether, and concentrated togive 650 mg (100% yield) of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, triethyl ester and characterized by:

¹ H NMR (CDCl₃) 8.2(d), 7.6(d), 4.3(q), 2.6-3.8(m), 1.55(t);

Fast atom bombardment mass spectrum [M+H]⁺ =588.

EXAMPLE 20

Preparation of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.

To a suspension of 200 mg (0.35 mmole) of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, triethyl ester (prepared by the procedure of Example 19) in 15 mlof water was added 59 μl of NaOH (50% wt/wt, 3 eq). The mixture wasstirred at 80° C. for 6 hours. The resulting homogeneous orange solutionwas then cooled to room temperature and freeze-dried to yield a brownsolid which was purified by HPLC (Q-Sepharose™, HPLC System III) using alinear gradient of 0-10% aqueous HOAc over 60 minutes to yield (50%) ofthe hydrolyzed product1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacticacid, and characterized by:

¹ H NMR (D₂ O) 8.22(d), 7.55(d), 3.60-3.90(m), 3.10-3.40(m).

EXAMPLE 21

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid; (EA-DO3A).

In a Parr-hydrogenater was placed 50 ml of an aqueous solution of 214 mg(0.43 mmole) of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 20) and 40 mg of 10% Pd/C.The suspension was shaken until hydrogen uptake ceased. Afterfiltration, the aqueous solution was freeze-dried yielding 197 mg (98%),as a tan solid,1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, and characterized by:

¹ H NMR (D₂ O) 7.35(d), 7.20(d), 3.10-3.70(m);

¹³ C NMR (D₂ O) 173.85, 172.50, 137.90, 131.90, 130.22, 121.55, 56.25,55.14, 54.42, 50.12, 49.65, 49.31, 31.00.

EXAMPLE 22

Preparation of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, 4,7,10-trimethyl ester.

To a stirred acetonitrile solution (20 ml) containing 1.0 g of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane (3 mmole)(prepared by the procedure of Example E) was added 1.6 g of methylbromoacetate (11 mmole) in one portion. After one hour potassiumcarbonate (0.3 g) was added and stirring continued at room temperaturefor twelve hours. The reaction mixture was then filtered and thefiltrate concentrated (in vacuo). The resulting residue was columnchromatographed (silica, CH₃ CN/CH₃ OH, 9:1, V:V) yielding the triesteras a white solid in 68 percent yield after concentration, and wasfurther characterized by:

¹ H NMR (CDCl₃) 8.19(d), 8.14(s), 7.18(d), 2.58-3.99(m);

¹³ C NMR (CDCl₃) 173.53, 172.83, 164.59, 142.28, 128.51, 127.69, 126.49,112.30, 57.25, 56.90, 55.03, 54.21, 53.06, 52.09, 52.00, 51.54, 50.68,50.30, 49.85, 49.63, 49.31, 49.00, 48.68, 48.37, 48.05, 47.70, 47.39.

EXAMPLE 23

Preparation of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.

A 6M hydrochloric acid solution (3 ml) containing 0.25 g of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, 4,7,10-trimethyl ester (0.45 mmole) (prepared by the procedure ofExample 22) was stirred and heated at 100° C. for 24 hours. Aftercooling to room temperature, 5 ml of water was added and the aqueoussolution freeze-dried to give a tan solid. Following columnchromatography (silica, Solvent System 5) the triacid was isolated in 60percent yield as an off-white solid, MP=228°-230° C. (dec), and furthercharacterized by:

¹ H NMR (D₂ O) 8.18(d), 8.09(s), 7.12(d), 3.87(s), 2.10-3.60(m);

¹³ C NMR (D₂ O) 180.45, 179.80, 163.66, 140.22, 128.60, 126.24, 122.66,111.50, 59.91, 59.06, 56.44, 52.75, 50.92, 48.84.

EXAMPLE 24

Preparation of1-(2-methoxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.

To a nitrogen purged aqueous (50 ml) solution containing 50 mg of PtO₂was added 50 mg of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 23). After hydrogenating in aParr bomb for one hour, the solution was filtered and the aqueousfiltrate freeze-dried to give the aniline derivative as a tan solid (95percent yield), MF>240° C. dec, and further characterized by:

¹ H NMR (D₂ O) 7.54(bs), 7.21(d), 7.09(d), 7.05(s), 6.88(s), 3.84(s),3.78(s), 2.85-3.56(m);

¹³ C NMR (D₂ O) 180.59, 179.77, 153.38, 124.16, 124.03, 122.82, 120.18,113.68, 112.43, 59.06, 57.02, 55.96, 53.67, 50.52, 50.08, 49.70, 49.04,48.32.

EXAMPLE 25

Preparation of1-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.

To an aqueous solution (1 ml) of1-(2-hydroxy-5-nitrobenzyl-1,4,7,10-tetraazacyclododecane (200 mg, 0.62mmole) (prepared by the procedure of Example F) was added bromoaceticacid (309 mg, 2.2 mmole, and NaOH (3.5 ml, ≈1M) with stirring at roomtemperature. The reaction progress was monitored by LC (anion exchange,Q-Sepharose™) and the pH maintained at about 8 via the addition of NaOHas needed. After 12 hours the solution was freeze-dried and the solidresidue chromatographed (column) on silica gel eluting with SolventSystem 5. The major yellow fraction was concentrated, dissolved in H₂ Oand freeze dried to give the desired product as a bright yellow powder(150 mg, 49 percent); MP=230°-235° C. (dec), and further characterizedby:

¹³ C NMR (D₂ O) 166.41, 165.60, 160.00, 119.91, 116.02, 113.85, 111.71,104.59, 45.20, 44.50, 43.96, 36.43.

EXAMPLE 26

Preparation of1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.

To an argon purged, aqueous solution (25 ml) ofN-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (200 mg, 0.4 mmole) (prepared by the procedure of Example 25) wasadded PtO₂ (130 mg) followed by the introduction of H₂ (Parrhydrogenator). After total disappearance of yellow color, the solutionwas purged with argon and filtered. The aqueous solution was thenfreeze-dried to give the product as a tan solid (146 mg, 78 percent).

EXAMPLE 27

Preparation ofa-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid, 1-isopropyl-4,7,10-trimethyl ester; (PN-DOTMA trimethylisopropylester).

To a solution of 1.00 g (2.37 mmole) ofa-[3-(4-nitrophenyl)propyl]-1,4,7,10-tetraazacyclododecane-1-aceticacid, 1-methyl ester (prepared by the procedure of Example P) in 35 mldry acetonitrile was added 1.15 g (8.32 mmoles) of anhydrous potassiumcarbonate with vigorous stirring under nitrogen. Optically activeα-benzene sulfonate of lactic acid methyl ester (1.79 g, 7.36 mmolesS:R=98.2) was added and the mixture was stirred at room temperature for60 hours. TLC analysis indicated formation of new products (R_(f) =0.71,Solvent System 2 for titled tetraesters and R_(f) =0.89 for triester).The resulting solution with suspended carbonate salts was poured into 40ml of chloroform and the precipitated inorganic salts were filtered.Solvent was removed from this filtrate and the crude orange oil waschromatographed twice on a 1 in.×16 in. flash silica gel column usingSolvent System 2 as the eluent to afford the titled product as anonresolved mixture of tetraester diastereomers (1.00 g, 1.47 mmole, 62%yield) which were free of underalkylated products.

¹ H NMR analysis of this material showed it to contain approximately 0.5equivalents of unreacted benzene sulfonate derivative. The NMR spectraof this material were not coalescent at 60° C. in chloroform:

¹ H NMR (CDCl₃, 60° C.) 7.9-8.1(m, 2H), 7.2-7.4(m, 2H), 5.06(m, 1H),3.4-4.0(m, 9H), 1.7-3.4(m, 20H), 1.0-1.7(m, 15H);

IR (CDCl₃) cm⁻¹ 2980, 2840, 1725, 1710 (ester carbonyl), 1590, 1510,1440, 1340;

Fast atom bombardment mass spectrum, m/e 702 [M+Na⁺ ]⁺, 687.

EXAMPLE 28

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-[tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid, 1-isopropyl-4,7,10-trimethyl ester; (PA-DOTMA trimethylisopropylester).

α-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid, 1-isopropyl-4,7,10-trimethyl ester (950 mg, 1.40 mmolesuncorrected) (prepared by the procedure of Example 27), which containedunreactedα-[3-(4-nitrophenyl)propyl]-1,4,7,10-tetraazacyclododecane-1-aceticacid, 1-methyl ester, was dissolved in 10 ml of 30% aqueous methanolcontaining 1.00 g of 10% palladium on carbon catalyst under a nitrogenatmosphere. Excess hydrogen was purged through the solution for 7 hours.At this point, TLC inspection revealed formation of a strongly ninhydrinpositive material (R_(f) =0.62 Solvent System 2, R_(f) =0.71 forstarting material). The methanolic catalyst slurry was filtered throughcelite and evaporation of solvent provided 800 mg of the crude titleproduct as a clear oil which contained benzene sulfonic acid (R_(f)=0.09 Solvent System 2) from the concomitant hydrogenolysis of thebenzene sulfonate ester. The crude oil was chromatographed on a 1 in.×8in. flash silica gel column with 10% methanol in chloroform which eluteda light yellow band in the void volume. Solvent System 2 was thenapplied to elute the titled product (480 mg) in 52% yield as a mixtureof unresolved diastereomers and geometric isomers. Three distinct abquartets were observed in the aromatic region of the ¹ H NMR:

¹ H NMR (CDCl₃, 3° C.) 6.63-8.0(m (quartets), 4H), 5.07(m, 1H, isopropylmethine H), 3.53-3.9(m, 13H with four singlets at 3.85, 3.73, 3.69 and3.56), 3.46(m, 1H), 3.25(broad t, 3H), 2-3.1(m, 14H), 1.0-2.0(m, 15H);

¹³ C NMR (CDCl₃, 30° C.) 177.3, 177, 176.4, 176.2, 175.9, 174.3, 172.9,170.6, 145.3, 144.9, 130.8, 129.7, 129.3, 129.1, 128.8, 128.4, 127.5,126.3, 115.2, 115.1, 114.9, 69.0, 68.4, 67.3, 61.6, 57.8, 57.4, 56.7,56.5, 56.4, 52.9, 52.7, 52.1, 50.8, 50.7, 50.6, 47.3, 47.1, 47.0, 46.9,46.5, 46.3, 46.1, 44.8, 44.5, 44.3, 34.1, 32.9, 32.5, 32.2, 31.8, 24.8,22.2, 22.1, 22.0, 21.8, 21.6, 15.8, 14.9, 7.7, 7.5, 7.4, 7.1;

IR (CDCl₃) cm⁻¹ 2960, 2840, 1720, 1510, 1450, 1375;

Fast atom bombardment mass spectrum, m/e 672 [M+Na⁺ ]⁺, 686.

EXAMPLE 29

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid; (PA-DOTMA).

The diastereomeric mixture ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid, 1-isopropyl-4,7,10-trimethyl ester (400 mg, 0.59 mmoles) (preparedby the procedure of Example 28) was dissolved in 50 ml of 6Nconcentrated hydrochloric acid and refluxed for 30 hours. TLC analysis(Solvent System 1) indicated conversion of ester (R_(f) =0.98 SolventSystem 1) to two new spots (R_(f) =0.6, high diastereomer and R_(f)=0.54, low diastereomer Solvent System 1, both spots UV, ninhydrin, andiodine positive). Excess solvent was removed on a rotary evaporator andafter drying, a mixture of crude diastereomers of the title product (403mg) was obtained as the hydrochloride salt. Preparative separation ofthe two diastereomers of the title product was accomplished by applying290 mg (0.51 mmole) of the crude salt to a 1×13 cm silica flash gelcolumn which was eluted with Solvent System 8. The high R_(f)diastereomer (119 mg, 0.21 mmole) was obtained in purer form than thelow R_(f) diastereomer (90 mg, 0.16 mmole) since high R_(f) diastereomereluted from the column first:

¹ H NMR for high R_(f) diastereomer:

¹ H NMR (D₂ O, 90° C., pD=7.5) 7.12(d, 2H), 6.80(d, 2H), 3.66(m, 1H),3.57(broad t, 3H), 2-3.4(m, 19H), 1.89(m, 1H), 1.37(m, 1H), 1.15(d, 3H),1.10(d, 3H), 1.06(d, 3H);

¹³ C NMR (D₂ O, 90° C., pD=7.5) 184.4, 184.2, 184.0, 183.3, 135.9,132.6, 131.7, 119.1, 78.2, 69.0, 61.7, 61.5, 61.1, 57.4, 54.1, 49.7,49.6, 48.2, 47.7, 47.2, 34.8, 33.8, 9.6, 9.1, 9.0;

Fast atom bombardment mass spectrum, m/e 566 [M+H⁺ ]⁺, 588 [M+Na⁺ ]⁺,604 [M+K^(+]) ⁺, 626 [M+K⁺ +Na⁺ -H⁺ ]⁺, 642 [M+2K⁺ -H⁺ ]⁺.

Preparation of Final Products--Complexes

Metal ligand complexes were prepared by various methods as shown below.The methods included mixing of the metal and ligand in aqueous solutionand adjusting the pH to the desired value. Complexation was done insolutions containing salts and/or buffers as well as water. Sometimesheated solutions were found to give higher complex yields than when thecomplexation was done at ambient temperatures. Also the work-up used inthe synthesis of BFC has been shown to effect the complexation.

EXAMPLE 30

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, ammonium salt, samarium(III) complex; Sm(BA-DOTA).

A small sample, 53 mg, (0.094 mmole) ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 3) was dissolved in 100 μl ofwater and treated with 3 ml of 0.043M Sm(OAc)₃ solution (48.8 mg, 0.128mmole) at pH 6-7. This solution was heated at 100° C. and the degree ofcomplexation was determined by anion exchange HPLC. When complexationwas complete by anion exchange chromatography, HPLC System III, thesolution of the complex was cooled to room temperature (about 25° C.)and freeze dried. The samarium complex was purified by silica gelchromatography (eluting with Solvent System 1). This procedure gave thetitle product in 82 percent yield. This complex was characterized byTLC, fast atom bombardment mass spectrometry, anion exchange HPLC, andfuther characterized by:

¹ H (D₂ O) 8.94, 7.81, 7.21, 6.97, 6.80, 5.63, 5.01, 4.58, 4.01, 3.56,1.78, 1.53, 1.24, 1.10, 0.77, -2.80, -2.95, -3.21, -3.91;

13C NMR (D₂ O) 190.3, 184.9, 181.4, 146.8, 134.3, 122.7, 116.4, 80.8,72.6, 64.2, 57.2, 55.8, 54.7, 53.4, 51.5, 49.7, 45.5, 43.5, 23.6.

Fast atom bombardment mass spectrum, [M+H⁺ ]⁺ =645 (Sm isotope pattern).

EXAMPLE 31

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

A solution of 150 μl Sm-153 (3×10⁻⁴ M, in 0.1N HCl) and 600 μl SmCl₃(3×10⁻⁴ M) in 0.1N HCl was prepared by adding the Sm-153 solution to theSmCl₃ solution. Then 5.0 μl of 2.0M NaOAc was added followed by 45 μl of50 mM ligand (prepared by the procedure of Example 3) in HEPES buffer.The pH of the solution was adjusted to 7 with 275 μl of 0.5M HEPES. Thissolution was split into two fractions of 500 μls each, and one fractionwas heated for 1 hour at 100° C.

The solutions were passed through SP-Sephadex™ cation excange resin. Thepercent of metal as a complex was determined using the procedurepreviously described. The results showed 99.6% of the metal as a complexfor the heated sample and 96.6% for the non-heated sample.

Stability of chelates by pH profile.

The stability of metal ligand complexes was measured by subjecting themto various pH values and analyzing for complex in solution. At low pH,protonation of the ligand can cause the release of metal. At high pH,metal hydroxides compete with chelate formation. Thus, when looking forinert chelates, a desirable inert chelate will remain as a chelate whenexposed to high and low pH values.

Profiles of the inertness at various pH values for some of the chelatesof this invention were generated using the methods described in thefollowing examples. In some cases non-chelated metal was removed bypassing the solution through a cation exchange resin. Dilute sodiumhydroxide and hydrochloric acid solutions were used to adjust the pH ofthe complex solutions from about 1 to about 14. The percent metal as acomplex was then determined by the methods previously described.

In a similar manner, several bifunctional chelate conjugates wereprepared and subjected to pH 2.8, 4.0 and 6.0. The amount of conjugateremaining in solution was determined by HPLC using a radiometricdetector. The results are shown in Example XX in the biology section.

EXAMPLE 32

pH Stability ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

The heated and non-heated samples from Example 30 were each split into2×250 μl aliquots. One 250 μl aliquot was adjusted with HCl to give thepH's represented in the table following. The second 250 μl aliquot wasadjusted with μl quantities of NaOH to the desired pH's. Once thedesired pH was reached, a 25 μl aliquot was removed and the percent ofmetal as complex determined as previously described. The results are asfollows:

    ______________________________________                % Complex                          Not    pH            Heated  heated    ______________________________________    1             98.9    92.7    3             99.6    93.3    5             99.6    92.9    7             99.6    95.7    9             99.9    95.8    11            99.6    95.7    13            99.6    94.5    ______________________________________

This data shows the stability for this sample whether heated or not andregardless of the pH tested.

EXAMPLE 33

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, yttrium(III) complex.

Ten μl of a solution containing 1.4 mg/100 μl ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 3) was added to 100 μl ofY(OAc)₃ (0.003M) spiked with Y-90. To this solution was added 500 μl ofwater followed by 125 μl of NaOAc (0.5M). Water (265 μl) was added tothe solution to bring the total volume to 1 ml. This solution wasdivided into two aliquots. One aliquot was heated to 100° C. for 1 hour.Both heated and non-heated samples were tested to determine the percentof metal as a complex using the cation exchange procedure previouslydescribed. The results showed 84% and 4% of the metal as a complex forthe heated sample and the non-heated sample, respectively.

EXAMPLE 34

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, lutetium(III) complex.

A solution ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 3) was dissolved in distilledwater to yield a concentration of 1.63 mg/ml.

A lutetium chloride solution was prepared by dissolving lutetiumchloride in 0.1N HCl to result in a concentration of 3.9 mg/ml (0.01M).Lutetium-177 (0.3 mmolar) was used as a tracer.

A volume of 30 μl of 0.01M lutetium solution was added to 2 μl of Lu-177solution. The appropriate amount of ligand from above was added andHEPES buffer (0.1M, pH=7.6) was added to give a total volume of 1.0 ml.The resultant solutions were 0.3 mmolar in ligand and lutetium. Thesolution was then heated to 100° C. for one hour and the percent of Luas a complex determined to be 86% by the cation exchange methoddescribed previously.

EXAMPLE 35

Preparation ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

A sample, 7 mg, (10.8 μmole) ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex (prepared by the procedure of Example 30)was dissolved in 400 μl water. Excess thiophosgene (50 μl) was added,followed by 400 μl CHCl₃ and the two-phase reaction stirred vigorouslyfor 30 minutes. At the end of this time, the water layer was extractedwith 500 μl CHCl₃ four times, and the water layer then was lyophilizedto give the desired titled product in quantitative yield.

The UV showed this compound to have a band at 272 and 282 nm. The TLC,silica gel developed by 75:25 V:V CH₃ CN:H₂ O, gave R_(f) =0.38. Thestarting material has an R_(f) =0.19. IR showed -SCN stretch at 2100cm⁻¹ ; fast atom bombardment mass spectrum [M+H⁺ ]⁺ =687.

EXAMPLE 36

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, ammonium salt, yttrium(III) complex.

A sample, 90 mg (160 mmole), ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 3) was dissolved in 1 mlwater. To this solution was added 3 ml of water containing 51 mg (192mmole) of Y(OAc)₃. The reaction mixture, pH=6 to 7, was then heated at100° C. for two hours. The resulting solution was then passed throughglass wool and lyophilized to give 126 mg of yellow solid. The solid waschromatographed on silica gel (Solvent System 1) to yield 71 mg (76percent) of the desired complex product. Analysis by anion exchangeshowed the same retention time as was found for the analogous Smcomplex. The title product was characterized by:

¹³ C NMR (D₂ O) 180.8, 180.5, 179.9, 146.1, 133.4, 122.0, 116.1, 74.9,66.3, 56.3, 55.8, 55.4, 55.1, 54.8, 52.2, 46.0, 44.0, 23.6;

¹ H NMR (D₂ O) 6.90(d), 6.70(d), 4.40(s), 3.45-3.06(m), 2.71-2.10(m),1.75(s).

Fast atom bombardment mass spectrum, [M+H⁺ ]⁺ =582.

EXAMPLE 37

Preparation ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, sodium salt, yttrium(III) complex.

A sample of theα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, yttrium complex (prepared by the procedure of Example 36) (10 mg,17 μmole) was dissolved in 400 μl H₂ O . To this solution was added 64μl thiophosgene (excess) and 400 μl CHCl₃ and the resulting mixturestirred vigorously for 40 minutes. During this time several smalladditions of solid NaHCO₃ were made to keep the pH at about 8. At theend of the reaction, the water layer was separated and extracted with 1ml of CHCl₃, four times, and lyophilized. The title product wascharacterized by TLC and UV spectroscopy.

EXAMPLE 38

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, yttrium(III) complex, ammonium salt; NH₄ [Y(PA-DOTA)].

The ligand,α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 8) was converted to the mixedprotonated-ammonium salt by chromatography on a strong cation exchangeresin (BP-Sephadex™ C-25, Pharmacia). The column was in the protonatedform and washed with distilled water. A concentrated aqueous solution ofthe ligand was applied to the column, followed by washing the columnwith distilled water. The ligand was eluted from the column with 0.5MNH₄ OH. The eluent was reduced to dryness.

A solution of Y(OAc)₃.4H₂ O (0.0728 g, 0.215 mmole) in 5 ml of distilledwater was added to a warm solution of the mixed protonated-ammonium saltligand (0.120 g, 0.215 mmole) in 5 ml of water. The solution was broughtto reflux and the pH adjusted to about 7.0 with 0.1M NH₄ OH. After 1hour at reflux, the solution was cooled and reduced to dryness. ExcessNH₄ OAc was removed by heating the white solid in an oil bath at about105° C. under vacuum. The title product (which contained about oneequivalent of ammonium acetate) yield was 0.142 g (94 percent) and wascharacterized by:

¹³ C NMR (D₂ O, 88° C., 75 MHz) 23.8, 27.6, 35.4, 49.6, 55.0, 56.2,56.9, 57.1, 65.7, 68.0, 121.7, 132.6, 138.8, 180.7, 181.4, 182.0, 183.1;

Fast atom bombardment mass spectrum, m/e 610 (positive ion, [M⁻ +2H⁺]⁺), 608 (negative ion, M⁻).

EXAMPLE 39

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex, ammonium salt; NH₄ [Sm(PA-DOTA)].

When the procedure of Example 38 was repeated, using Sm(OAc)₃.3H₂ O(0.082 g, 0.215 mmole) in place of Y(OAc)₃.4H₂ O , the title product(which contained about one equivalent of ammonium acetate) was preparedin a yield of 0.155 g (94 percent) and was characterized by:

¹³ C NMR (D₂ O, 88° C., 75 MHz) 23.5, 27.2, 35.6, 50.5, 54.5, 56.0,57.2, 57.9, 69.5, 71.5, 122.3, 133.0, 139.7, 181.2, 188.4, 189.3, 190.9;

Fast atom bombardment mass spectrum, m/e 673 (positive ion, [M⁻ +2H⁺ ]⁺,Sm isotope pattern), m/e 671 (negative ion, M⁻, Sm isotope pattern).

EXAMPLE 40

Preparation ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex, ammonium salt; NH₄ [Sm(SCN-PA-DOTA)].

Solutions of Sm(OAc)₃.3H₂ O (27.9 mg, 381.56 g/mole, 73.1 μmole) in 10ml of distilled water and 42 mg of PA-DOTA, mixed ammonium-potassiumsalt (632.97 g/mole, 66.4 μmole) (prepared by the procedure of Example8) in 10 ml of distilled water were mixed and heated on a steam bath.After 30 min., the reaction to form [Sm(PA-DOTA)] was complete asdetermined by HPLC using the method described in Example 41. Thesolution was reacted with a solution of 45.4 mg of CSCl₂ (114.98 g/mole,395 μmole) in 20 ml of chloroform by shaking in a separatory funnel. Thereaction was complete after about one min. as determined by HPLC and bythe absence of a positive test with ninhydrin (0.2% in ethanol) when theaqueous solution was spotted on a silica gel TLC plate (the startingchelate, [Sm(PA-DOTA)], tested positive). The chloroform layer wasremoved and the aqueous layer was washed with two 20 ml portions ofchloroform. The aqueous layer was reduced to dryness by the addition of100 ml of acetonitrile and evaporated at ambient temperature under astream of nitrogen to yield 56 mg of the title product.

Fast atom bombardment mass spectrum, m/e 715 (positive ion, [M⁻ +2H⁺ ]⁺,Sm isotope pattern), m/e 713 (negative ion, M⁻, Sm isotope pattern).

EXAMPLE 41

HPLC Analysis of the rate of chelation of Y³⁺ with PA-DOTA.

The rate of PA-DOTA chelation with Y³⁺ was studied as a function of thework-up used in the synthesis of PA-DOTA. The extent of chelation wasmonitored by HPLC using an Alltech Econosphere™ C18 100 mm column. Thegradient used was: A) 95:5 of pH 6.0 0.05M NaOAc buffer:CH₃ CN, B) 30:70of pH 6.0, 0.05M NaOAc buffer:CH₃ CN, A to B in 15 min. A solution ofPA-DOTA, hydrochloride salt, (retention time=1.31 min) and of PA-DOTAmixed ammonium-potassium salt (retention time=4.55 min), both preparedby the procedure of Example 8, were prepared as 1.6 mM, pH 6.0 0.5MNaOAc buffer. One ml (1.6 μmole) of each of the two solutions wasreacted with 0.2 ml (8 μmole) of Y(OAc)₃.4H₂ O solution (40 mM in pH 6.00.5M NaOAc buffer). Chelate formation was determined by the appearanceof a peak at a retention time=3.96 min (confirmed by comparison to asample from Example 38). Chelate formation results were as follows.

    ______________________________________    PA-DOTA Salt  % Complex   Time(min)/Temp.    ______________________________________    PA-DOTA, HCl salt                  >99         <1/room temp.    PA-DOTA mixed <10         15/room temp.    ammonium-potassium                  >85         10/90° C.    salt    ______________________________________

Clearly, the method of purification of the BFC has an effect on the rateof chelation.

EXAMPLE 42

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(methylacetic)acid, samarium(III) complex, ammonium salt; NH₄ [Sm(PA-DOTMA)].

A solution of Sm(OAc)₃.3H₂ O (13.2 mg in 2 ml of distilled water) wasadded to a solution ofa-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(methylacetic)acid (30 mg in 2 ml of distilled water), (prepared by the procedure ofExample 29, high R_(f) diastereomer). The pH 6.5 solution was heated ona steam bath and the reaction was monitored by HPLC. After 30 min. ofheating the solution was reduced to dryness on a rotary evaporator anddried in a vacuum oven.

Fast atom bombardment mass spectrum, m/e 715 (positive ion, [M+2H⁺ ]⁺,Sm isotope pattern), 737 (positive ion, [M+H⁺ +Na⁺ ]⁺, Sm isotopepattern) 713 (negative ion, (M⁻), Sm isotope pattern).

In the following examples the complex solutions were passed through anion exchange column to remove any excess free metal from solution.Labile systems would return to equilibrium and similiar amounts of thefree metal would be in solution. Inert systems would not re-equilibrateand the amount of non-chelated metal should decrease. Thus even though acomplex can be formed in low yield, a purification by passing it throughan ion exchange resin could result in a usable stable complex withoutuncomplexed metal.

EXAMPLE 43

Preparation ofα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

A 3×10⁻² M solution ofα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 12) was prepared bydissolving 5.8 mg in 325 μl NANOpure™ water. A 10 μl aliquot of thissolution was added to 990 μl of 3×10⁻⁴ M SmCl₃ (in 0.1N HCl) spiked withSm-153. The pH was then brought to 7.0 with NaOH. This solution wassplit into two fractions of 500 μl each, and one fraction was heated for1 hour at 100° C. Both heated and non-heated samples were tested todetermine the percent of metal as a complex by the cation exchangemethod described previously. The solution was then passed throughSP-Sephadex™ resin. Complexation was again determined as the purifiedsamples. The results are shown in the following table.

    ______________________________________           Heated            Non-Heated                 Not                 Not           Purified                 Purified    Purified                                     Purified    ______________________________________    %        96      95          97    89    Complex    ______________________________________

EXAMPLE 44

pH Stability ofα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

The heated and non-heated purified samples from Example 43 were eachsplit into 2×250 μl aliquots. One aliquot was adjusted with HCl and theother with NaOH to give the pH's represented in the table following.After the desired pH was reached the sample was allowed to stand for 10min and the amount of metal as a complex was determined by the cationexchange method described previously. The results are shown in thefollowing table.

    ______________________________________                % Complex                          Not    pH            Heated  heated    ______________________________________    2             95      94    3             97      95    5             98      97    7             96      97    9             98      96    11            97      98    13            100     99    ______________________________________

EXAMPLE 45

Preparation ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium(III) complex.

A 3×10⁻² M solution ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (prepared by the procedure of Example 11) was prepared bydissolving 7.9 mg in 464 μl NANOpure™ water. A 10 μl aliquot of thissolution was added to 950 μl of 3×10⁻⁴ M SmCl₃ (in 0.1N HCl) spiked withSm-153. The pH was then brought to 7.0 with NaOH. This solution wassplit into two fractions of 500 μl each, and one fraction was heated for1 hour at 100° C. Both heated and non-heated samples were tested todetermine the percent of metal as a complex using the cation exchangemethod described previously. The solutions were passed throughSP-Sephadex™ resin. Complexation was then determined on the purifiedsamples using the general procedure described hereinbefore. The resultsare shown in the following table.

    ______________________________________            Heated            Non-Heated                  Not                 Not            Purified                  Purified    Purified                                      Purified    ______________________________________    % Complex 100     72          100   46    ______________________________________

EXAMPLE 46

pH Stability ofα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, Sm(III) complex.

The heated and non-heated purified sample from Example 45 were eachdivided into two aliquots. One aliquot was adjusted with dilute HCl andthe other with NaOH to give the pH's represented in the table following.After the desired pH was reached the sample was allowed to stand for 10min and the amount of metal as a complex was determined by the cationexchange method described previously. The results are shown in thefollowing table:

    ______________________________________                  Purified                           No    pH              Heat   heat    ______________________________________    2               99     99    4               100    100    5               100    100    7               100    100    9               100    100    11              100    100    13              100    100    ______________________________________

EXAMPLE 47

Preparation ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium(III) complex.

A 0.0219M (100 μl, 2.195 mmole) solution ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid(prepared by the procedure of Example 4) was contacted with a 0.043M (25μl, 1.075 mmole) solution of Sm(OAc)₃. The reaction mixture wassubjected to anion exchange HPLC (Q-Sepharose™ column, 1 cm×25 cm, flowrate=2 ml/min, eluted with 0-1M NH₄ OAc gradient over 30 min). After onehour, the complex (retention time=11.1 min) had formed in 77 percentyield (area percent) and was completely resolved from the ligand peak(retention time=15.5 min). Additional evidence that the title complexhad formed was obtained on silica gel TLC (Solvent System 4) whereby theligand has an R_(f) =0.59 and the complex has an R_(f) =0.00.

EXAMPLE 48

Preparation ofα-(4-aminophenyl)-1.4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium(III) complex andα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid,samarium(III) complex.

A solution, 0.022M (100 μl, 2.2 μmole), of the isomers (prepared by theprocedure of Example 5) was contacted with a 0.043M (6.4 μl, 0.275μmole) solution of Sm(OAc)₃. The reaction mixture was subjected to anionexchange HPLC (Q-Sepharose™ column, 1 cm×25 cm, flow rate=2 ml/min,eluted with 0-0.25M NH₄ OAC gradient over 30 min). After forty minutes,the complex (retention time=9.0 min) had formed in 66 percent yield(area percent) and was completely resolved from the ligand peak(retention time=18.5 min).

EXAMPLE 49

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium-153 complex andα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid,samarium-153 complex.

A 3×10⁻² M solution ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidpentahydrochloride andα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acidpentachloride (prepared by the procedure of Example 5) was prepared bydissolving 4.2 mg in 300 μl NANOpure™ water. A 10 μl aliquot of thissolution was added to 15 μl of 2×10⁻² M SmCl₃ (in 0.1N HCl) spiked withSm-153. The volume was brought to 1 ml by addition of water. The pH wasthen brought to 7.0 with NaOH. This solution was split into twofractions of 500 μl each, and one fraction was heated for 1 hour at 100°C.

Both heated and non-heated samples were tested to determine the percentof metal as a complex by the cation exchange method describedpreviously. The results showed 89% and 96% of the metal as a complex forthe heated and non-heated samples, respectively.

EXAMPLE 50

pH Stability ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium(III) complex, andα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid,samarium(III) complex.

The heated sample from Example 49 and the non-heated sample were eachdivided into 2 aliquots. One aliquot was adjusted with HCl and the otherwith NaOH to give the pH's represented in the table following. Once thedesired pH was reached, the samples were allowed to stand for 10 minutesand the percent of metal as complex was determined by the cationexchange method described previously. The results are shown in thefollowing table:

    ______________________________________                % Complex                          Not    pH            Heated  heated    ______________________________________    1             86      83    3             99      99    5             99      99    7             89      96    9             99      98    11            96      89    13            97      97    ______________________________________

EXAMPLE 51

Preparation ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium(III) complex.

Thirteen microliters of 2×10⁻² M solution ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid(prepared by the procedure of Example 4) 15 μl of SmCl₃ (0.02M 0.1N HCl)and 2 μl of Sm-153, had the volume brought to 1 ml with NANOpure™ water.The pH was adjusted to 7.0 with NaOH. This solution was split into twofractions of 500 μl each, and one fraction was heated for 1 hour at 100°C. Both heated and non-heated samples were tested to determine thepercent of metal as a complex using the cation exchange proceduredescribed previously. The results showed 74% and 42% of the metal as acomplex for the heated and non-heated samples, respectively.

EXAMPLE 52

pH Stability ofα-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,samarium(III) complex.

The heated sample of Example 51 was divided into 2 aliquots. One aliquotwas adjusted with HCl and the other with NaOH to give the pH valuesrepresented in the following table. After the desired pH was reached thesample was allowed to stand for 10 minutes and the amount of metal as acomplex was determined by the cation exchange method describedpreviously. The results are shown in the following table.

    ______________________________________           pH   % Complex    ______________________________________           1    67           2    70           4    70           5    72           7    74           9    76           10   76           12   75           13   76    ______________________________________

EXAMPLE 53

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, lutetium(III) complex.

A solution of EA-DO3A (prepared by the procedure of Example 17) was madeby dissolving the solid in water. The final concentration was O.01M.

A lutetium chloride solution was prepared by dissolving lutetiumchloride in 0.1N HCl. The resultant concentration was 3.9 mg/ml oflutetium chloride (0.01M Lu). Lutetium-177 was used as the tracer.

A volume of 30 μl of 0.01M Lu solution was added to 2 μl of Lu-177solution. A volume of 30 μl of ligand was added and HEPES buffer (0.1M,pH=7.6) was added to give a total volume of 1.0 ml. The resultantsolution was 0.3 mM in ligand and lutetium.

The amount of Lu as a complex was determined to be 98 percent by thecation exchange method previously described.

EXAMPLE 54

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacylcododecane-4,7,10-triaceticacid, yttrium (III) complex; [Y(EA-DO3A)]

The ligand,1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 17) was converted to themixed protonated-ammonium salt by chromatography on a strong cationexchange resin (SP-Sephadex™ C-25, Pharmacia). The column was in theprotonated form and washed with distilled water. A concentrated aqueoussolution of the ligand was applied to the column, followed by washingthe column with distilled water. The ligand was eluted from the columnwith 0.5M NH₄ OH. The eluate was reduced to dryness on a rotaryevaporator and dried in a vacuum oven.

A solution of Y(OAc)₃.4H₂ O (0.203 g, 338.101 g/mole, 0.600 mmole) in 10ml of distilled water was added to a warm solution of the mixedprotonated-ammonium salt ligand (0.300 g, 499.615 g/mole, 0.600 mmole)in 10 ml of distilled water. The solution was brought to reflux and thepH was adjusted to about 7.0 with 0.1M NH₄ OH. After 15 minutes atreflux, the solution was cooled and reduced to dryness on a rotaryevaporator. Excess ammonium acetate was removed by heating the whitesolid in an oil bath at about 105° C. under vacuum. The title product(which contained about one equivalent of ammonium acetate) was providedin a yield of 373 mg (99 percent) and was characterized by:

¹³ C NMR (88° C., D₂ O, 75 MHz) 24.4, 28.4, 51.2, 56.5, 57.2 (2C), 57.7,67.2, 67.6, 120.5, 132.3, 135.2, 182.1, 182.5, 183.0;

Fast atom bombardment mass spectrum, m/e 552 (positive ion, [M+H⁺ ]⁺),m/e 610 (negative ion, [M+OAc⁻ ]⁻).

EXAMPLE 55

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium complex; [Sm(EA-DO3A)].

When the procedure of Example 54 was repeated, using Sm(OAc)₃.3H₂ O(0.229 g, 381.531 g/mole, 0.600 mmole) in place of the Y(OAc)₃.4H₂ O ,the title product (which contained about one equivalent of ammoniumacetate) was prepared in a yield of 408 mg (99 percent) and wascharacterized by:

¹³ C NMR (88° C., D₂ O, 75 MHz) 23.8, 27.9, 51.4, 57.5, 58.1 (3C), 68.7,73.4, 120.3, 132.2, 134.7, 185.0. 186.8, 192.1;

Fast atom bombardment mass spectrum, m/e 615 (positive ion [M+H⁺ ]⁺, Smisotope pattern), m/e 673 (negative ion, [M+OAc⁻ ]⁻, Sm isotopepattern).

EXAMPLE 56

Preparation of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

Ten microliters of 1.48 mg/100 μl (0.03M) solution of1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-tetraaceticacid (prepared by the procedure of Example 20) was added to 15 μl ofSmCl₃ solution (0.02M in 0.1N HCl) and spiked with Sm-153. The volumewas brought to 1 ml with NANOpure™ water. This solution was divided intotwo 500 μl fractions, and one fraction was heated for 1 hour at 100° C.Both heated and non-heated samples were tested to determine the percentof metal as a complex using the cation exchange procedure describedhereinbefore. The results showed 81% and 43% of the metal as a complexfor the heated and non-heated samples, respectively.

EXAMPLE 57

pH Stability1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

The heated sample of Example 56 was divided into two aliquots. Onealiquot was adjusted with HCl and the other with NaOH to give the pH'srepresented in the following table. After the desired pH was reached thesample was allowed to stand for 10 min and the amount of metal as acomplex was determined by the cation exchange method describedpreviously. The results are shown in the following table.

    ______________________________________           pH   % Complex    ______________________________________           1    20           2    37           4    49           5    58           7    81           9    62           10   64           12   64           13   54    ______________________________________

EXAMPLE 58

Preparation of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

Three μl of 4.65 mg/100 ml solution of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 20) was added to a solutionof 15 μl of SmCl₃ (0.02M in 0.1N HCl) previously spiked with Sm-153. Thevolume was brought to 1 ml with NANOpure™ water. The pH of the solutionwas adjusted to 7.3 using NaOH. The solution was divided into twoaliquots, and one aliquot was heated at 100° C. for 1 hour. Both heatedand non-heated samples were tested to determine the percent of metal asa complex by the cation exchange method described previously. Theresults showed 96% and 93% of the metal as a complex for the heated andnon-heated samples, respectivly.

EXAMPLE 59

pH Stability of1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

The heated and non-heated samples of Example 58 were each divided intotwo aliquots. One aliquot of each sample was adjusted with HCl and theother with NaOH to give the pH's shown in the following table. After thedesired pH was reached, the sample was allowed to stand for 10 minutesand the amount of metal as a complex was determined by the cationexchange method described previously. The results are shown in thefollowing table.

    ______________________________________                % Complex    pH            Heated  Not Heated    ______________________________________    1             44      13    3             50      42    5             73      55    7             96      93    9             98      98    11            89      90    13            28      37    ______________________________________

EXAMPLE 60

Preparation of1-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

A 3×10⁻² M solution of1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 25) was prepared bydissolving 3.9 mg in 260 μl NANOpure™ water. A 10 μl aliquot of thissolution was added to 15 μl of 2×10⁻² M SmCl₃ (in H₂ O ) spiked with 10μl of Sm-153 in 0.1N HCl (3×10⁻⁴ M). The volume was brought to onemilliliter by addition of 965 μl DI H₂ O . The pH was then brought to7.0 with NaOH. This solution was split into two fractions of 500 μleach, and one fraction was heated for 1 hour at 100° C. Both heated andnon-heated samples were tested to determine the percent of metal as acomplex by the previously described cation exchange method. The resultsshowed 71 percent and 74 percent of the metal as a complex for theheated and non-heated samples, respectively.

EXAMPLE 61

pH Stability of1-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

The heated and non-heated sample of Example 60 were each split into two250 μl aliquots. One aliquot was adjusted with HCl and the other withNaOH to give the pH's represented in the table following. After thedesired pH was reached the sample was allowed to stand for 10 minutesand the amount of metal as a complex was determined by the cationexchange method described previously. The results are shown in thefollowing table.

    ______________________________________                % Complex                          Not    pH            Heated  heated    ______________________________________    1             65      51    3             98      93    5             99      99    7             100     100    9             100     99    11            100     96    13            100     98    ______________________________________

EXAMPLE 62

Preparation of1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

A 3×10⁻² M solution of1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid (prepared by the procedure of Example 26) was prepared bydissolving 3.2 mg in 100 μl NANOpure™ water. A 10 μl aliquot of thissolution (diluted to 20 μl) was added to 15 μl of 2×10⁻² M SmCl₃ (in H₂O) spiked with Sm-153. The volume was brought to 1 ml by addition of 950μl DI H₂ O and 20 μl 0.1N NaOH. This solution was split into twofractions of 500 μl each, and one fraction was heated for 1 hour at 100°C. Both heated and non-heated samples, were tested to determine thepercent of metal as a complex by the cation exchange method describedpreviously. When less than 95 percent of the metal was complexed, thesolution was passed through SP-Sephadex™ resin. The percent of metal asa complex was again determined. The results are shown in the followingtable.

    ______________________________________           Heated            Non-Heated                 Not                 Not           Purified                 Purified    Purified                                     Purified    ______________________________________    %        99      68          99    21    Complex    ______________________________________

EXAMPLE 63

pH Stability of1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium(III) complex.

The heated and non-heated purified chromatographed samples from Example62 were each split into two 250 μl aliquots. One 250 μl aliquot wasadjusted with HCl and the other with NaOH to give the pH's representedin the table following. Once the desired pH was reached, samples wereallowed to stand for 10 minutes and the percent of metal as complex wasdetermined by the cation exchange method described previously. Theresults are as shown in the following table:

    ______________________________________                % Complex                          Not    pH            Heated  heated    ______________________________________    1             60      56    2             74      76    3             84      87    5             97      98    7             99      99    9             99      98    11            99      99    13            99      99    ______________________________________

Methods of Use-Biology

Bifunctional chelants having within the same molecule a metal chelatinggroup such as DTPA and a reactive linker (the aryl amine) are known tobe capable of being covalently attached to various target directedbiomolecules having specificity for cancer or tumor cell epitopes orantigens. Radionuclide complexes of such conjugates are useful indiagnostic and/or therapeutic applications as means of conveying theradionuclide to a cancer or tumor cell. [Reference, Meares et al., Anal.Biochem. 142, 68-78 (1984); see also discussion on pp. 215-216 of J.Protein Chem., 3(2) (1984) by Meares and Goodwin, more recent referencesare Meares, U.S. Pat. No. 4,678,677, issued Jul. 7, 1987, Warshawsky etal. U.S. Pat. No. 4,652,519, issued Mar. 24, 1987 and Brechbiel, et al.,Inorg. Chem., 25, 2772-2781 (1986)]. Conceivably, the product, employingradioactive or luminescent metals, may also be used for in vitroimmunoassays.

Background Information

The utility of the labeled antibodies depends on a number of factors,for example: 1) the specificity of the antibody, 2) the inertness orstability of the complex under use conditions (i.e., serum stability),and 3) the integrity of the antibody, i.e. the specificity and theimmunoreactivity of the antibody, is not affected by the labelingprocess.

Stability or inertness of the complex is of utmost importance to theeffectiveness of the radionuclide-antibody conjugate as a diagnosticand/or therapeutic agent. Kinetic lability can lead to instability, e.g.in the serum, and the dissociation of the radionuclide from the complex.Thus it diminishes the diagnostic and therapeutic effectiveness. Inaddition, it poses a greater potential for general radiation damage tonormal tissue. [Cole, et al., J. Nucl. Med., 28, 83-90 (1987)].

Linking of Radionuclide to Antibody

Attachment of radionuclide to antibody can be carried out by eitherlinking the BFC to the antibody via procedures well known in the art,followed by chelation of the radionuclide under conditions compatiblewith the antibody, or alternatively, conjugation of the antibody topreformed (ambient or elevated temperature) metal--BFC complex. [Meareset al., Acc. Chem. Res. 17, 202-209 (1984)]. Examples are provided.

EXAMPLE ZA (Comparative)

Conjugation of 1-(4-isothiocyanatobenzyl)-diethylenetriaminepentaaceticacid, samarium-153 complex to IgG and F(ab')₂ of CC-49; [¹⁵³Sm(SCN-Bz-DTPA)]-IgG and [¹⁵³ Sm(SCN-Bz-DTPA)]-F(ab')₂ fragment.

The 1-(4-isothiocyanotobenzyl)diethylenetriaminepentaacetic acid,samarium-153 complex [¹⁵³ Sm(SCN-Bz-DTPA)] was prepared by mixing 150 μl¹⁵³ Sm in 0.1N HCl with 9 μl of1-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid to whichwas added HEPES buffer (0.5M, pH 8.9, approximately 30 μl) to bring thepH to about 6. To conjugate, 22.5×10⁻⁹ moles of IgG or F(ab')₂ of CC-49(about 1×10⁻⁴ M concentration of protein in 50 mmole HEPES, pH 8.5) wasmixed with the ¹⁵³ Sm complex, and the pH was adjusted to 8.9 byaddition of a sodium carbonate solution (1.0M, 12-15 μl). Conjugationwas carried out at room temperature (about 25° C.) for about 3 hours.The ¹⁵³ Sm complex labeled IgG or F(ab')₂ was isolated and characterizedsimilarly as described in Example XII.

Experimental for Biology

EXAMPLES I, & II and Comparative Examples A-D

IN VIVO SCREENING OF BIFUNCTIONAL CHELATES.

The stability of certain rare earth chelates has been examined by invivo testing in animals. For example, Rosoff, et al. in theInternational Journal of Applied Radiation and Isotopes 14, 129-135(1963) report on the distribution of radioactive rare earth chelates inmice for certain aminocarboxylic acids. It was found that in vivo "thecompetition between the chelating agent and body constituents (inorganicand organic) for the rare-earth ions, determines its deposition andexcretion." The strong rare-earth chelates of this invention arebelieved to dissociate very little and be excreted, while the weak andintermediate strength chelates known in the art dissociate more readilyand thus are deposited in organs such as the liver. However,concentration of radionuclide in the liver is not always due to weakcomplex formation, but in some cases is due to the affinity that themetal chelate has for the liver. Chelates have, in fact, been preparedand utilized for the evaluation of liver function [Fritzberg, Alan R.,Radiopharmaceuticals: Progress and Clinical Perspectives, Vol. 1, 1986;U.S. Pat. Nos. 4,088,747 and 4,091,088].

The biodistribution of the yttrium and samarium chelates of the compoundof Example 3, an example of a strong rare-earth chelate, were determinedand the percent dose in the liver was used as an in vivo screeningprocedure to qualitatively estimate the stability of the chelates.Chelates of NTA and EDTA are included for comparison. Also samarium wasinjected as samarium chloride in unchelated form as a control.

Sprague-Dawley rats weighing from 150 to 200 g were purchased fromCharles River Laboratories. These animals were placed in cages and fedwater and food ad libitum. The animals were acclimated for at least fivedays before use. Prior to injection of complex, the animals were placedunder a heat lamp (15 to 30 minutes) to dilate the tail vein. The animalwas then placed in a restraining cage, the tail cleaned with an alcoholswipe, and the animal injected (50 to 200 μl) via the tail vein. Afterinjection, the animal was placed in another cage for two hours afterwhich time the animal was sacrificed by cervical dislocation. The animalwas then dissected, the parts rinsed with deionized H₂ O, patted dry,and weighed into a tared counting vial. At least three standards of thesame material as injected were prepared and counted with the animaltissues. Percent of dose is the number of counts in the organ divided bythe number of counts in the standard multiplied by 100 (see thefollowing Table).

                  TABLE    ______________________________________    Biodistribution Data                                   % Injected    Example  Ligand of             Dose in    No.      Ex.*           Metal  Liver    ______________________________________    I        3              Y      0.17    II       3              Sm     0.29    (A)      EDTA           Sm     8.4    (B)      EDTA           Sm     4.4    (C)      NTA            Sm     8.6    (D)      SmCl.sub.3     Sm     39    ______________________________________     *Complexes were prepared at ligand/metal ratios of 1:1 for Examples I and     II; at 5:1 for Example A; and at about 300:1 for Examples B and C.

EXAMPLES III AND E

The 1:1 complexes of yttrium (spiked with a tracer amount of ⁹⁰ Y) withthe ligand of Example 3, which is BA-DOTA, and EDTA (Comparative E) wereprepared by methods described previously. Several 100 μL aliquots werethen transferred to separate centrifuge tubes. Excess Y(III) was addedsuch that the total volume change is minimized and the time noted.One-half hour after metal addition, the percent complex was determinedby the cation exchange method described previously and this was comparedto the original amount of complex. The percent complex versus addedmetal gives an indication as to the lability of the ligand-metalcomplex. The results are given in the Table.

                  TABLE    ______________________________________    Complex Study    Metal/Ligand    % Complex    Molar Ratio     BA-DOTA   EDTA    ______________________________________     1              80        98     10             --        86    100             --        78    250             88        48    500             80        16    ______________________________________

EXAMPLE IV

Preparation of1-[2-(4-isothiocyanatophenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium-153 complex; [¹⁵³ Sm(SCN-EA-DO3A)] complex.

To 100 μl of ¹⁵³ Sm in 0.1N HCl was added 5 μl of SCN-EA-DO3A (5mM in 50mM HEPES, pH 8.2) (prepared in Example 18). This was mixed on a vortexmixer, and HEPES buffer (0.5M, pH 8.9) was added gradually (about 25 μltotal) to adjust the pH to around 7. The progress of the chelation wasmonitored by HPLC on GF-250 column (HPLC System I). Yields around 50%were obtained.

EXAMPLE V

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex; [¹⁵³ Sm(PA-DOTA)] complex.

To 150 microliters (μl) of ¹⁵³ Sm solution in 0.1N HCl (approximately4.6 mCi) were added 1 μl of sodium acetate (2.0M) and 9 μl ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid mixed ammonium, potassium salt (5 mmole in 50 mmole HEPES, pH 8.5,prepared by the procedure of Example 9). The mixture was mechanicallyshaken and titrated gradually with a HEPES buffer (0.5M, pH 8.9;approximately 31 μl added) to pH 7. This was then heated at 98° C. in asand bath for 1 hour. Upon termination, 5 μl of the mixture was used foranalysis on a Mono-Q™ column on HPLC System II, eluted with a gradientsolvent system (0-15 minutes, from 0 to 100% B; where A=water, andB=1.0M ammonium acetate and 0.1 mmole EDTA). Yields of 85-95% based on¹⁵³ Sm were obtained. Theα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex thus prepared was characterized by comparisonwith the identical non-radioactive sample, by their respectivechromatographic behavior on Mono-Q™ and GF-250 columns. Further evidenceof the presence of the radioactive complex was determined by theconversion to the isothiocyanato derivative and its subsequentconjugation to antibody. The complex has also been prepared withoutheating (at ambient temperature) by incubation for 6 to 18 hours toresult in 70-80% yield.

EXAMPLE VI

Preparation ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7-10-tetraazacyclododecane-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex; [¹⁵³ Sm(SCN-PA-DOTA)] complex.

To the reaction mixture obtained in Example V was added 2 μl of HEPESbuffer (0.5M, pH 8.9), 2 μl of thiophosgene and 0.2 ml of chloroform.The mixture was mechanically shaken vigorously 2 or 3 times for a fewseconds each time. The chloroform layer was discarded and the aqueouslayer which contained mainly the desired product was saved and furtherpurified. The yield ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex, based on ¹⁵³ Sm activity measurement by HPLCon GF-250 column using HPLC System I, was 85-90%. To purify, the aqueouslayer was passed through a Sep-Pak™ C-18 cartridge and eluted with 90%acetonitrile in water. The first 300 μl of effluent was discarded, andthe SCN-derivative which came off in the next 900 μl was characterizedby HPLC on GF-250. The recovery of the ¹⁵³ Sm activity was better than90%. The bulk of the solvent was then evaporated over a period of 1.5 to2 hours, and the residue was used for conjugation to antibody.

EXAMPLE VII

Preparation ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex; [¹⁵³ Sm (BA-DOTA)] complex.

To 200 μl of ¹⁵³ Sm solution in 0.1N HCl were added 1 μl of sodiumacetate (2.0M) and 12 μl ofα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (5 mmole in 50 mmole HEPES, pH 8.5) (prepared by the procedure ofExample 3). The mixture was mechanically shaken and titrated graduallywith a HEPES buffer (0.5M, pH 8.9; approximately 38 μl added) to pH 7.This was then heated at 98° C. in a sand bath for 1 hour. Upontermination, 5 μl of the mixture was used for analysis on a Mono-Q™column (HPLC System II, eluted with a gradient solvent system: 0-15minutes, from 0 to 100% B; where A=water, and B=1.0M ammonium acetateand 0.1 mmole EDTA). In general, yields of 85-95% based on ¹⁵³ Sm wereobtained. The complex has also been prepared by incubation of themixture at room temperature for 12-18 hours which resulted in a yield ofthe title product of 80-90%. Theα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex thus prepared was characterized by comparisonwith an authentic sample by their chromatographic behavior on theMono-Q™ column, conversion to the isothiocyanato derivative and itssubsequent conjugation to antibody.

EXAMPLE VIII

Preparation ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex; [¹⁵³ Sm(SCN-BA-DOTA)] complex.

To the reaction mixture obtained in Example VII were added 2 μl of HEPESbuffer (0.5M, pH 8.9), 2 μl of thiophosgene and 0.2 ml of chloroform. Itwas mechanically shaken vigorously 2 or 3 times for a few seconds eachtime. The chloroform layer was discarded and the aqueous layer whichcontained mainly the desired product was saved and further purified. Theyield ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex, as analyzed by HPLC on GF-250 column basedon the ¹⁵³ Sm activity using the HPLC System I was over 90%. To purify,the aqueous layer was passed through a Sep-Pak™ C-18 cartridge andeluted with 90 percent acetonitrile in water. The first 0.3 ml ofeffluent was discarded, and the desired product came off in the next 1.2ml, with 86-93% recovery. The bulk of the solvent was then evaporatedover a period of about 2 hours, and the residue was used for conjugationto antibody.

EXAMPLE IX

Conjugation of1-[2-(4-isothiocyantophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samariuim-153 complex to antibody; [¹⁵³ Sm(SCN-EA-DO3A)]-IgGconjugate.

The antibody used was CC-49, a murine monoclonal IgG that binds to anepitope of TAG-72, a tumor associated antigen. To conjugate, 187 μl ofthe antibody solution (1.20×10⁻⁴ M in 50 mM HEPES, pH 8.5) was mixedwith 4.5×10⁻⁸ moles of ¹⁵³ Sm(SCN-EA-DO3A) prepared as described inExample IV followed by addition of a sodium carbonate solution (1.0M) toraise the pH to 8.9. The reaction was allowed to continue for 2 hours atroom temperature. Upon termination, the ¹⁵³ Sm labeled IgG was isolatedby centrifugal gel filtration on Sephadex G-25 (2.2 ml) disposablecolumns, and further purified by HPLC on GF-250 column, eluted with acitrate buffer (0.25M, pH 7.4). The fractions contained the labeled IgGwere pooled, concentrated and exchanged (3×) into PBS by use ofCentricon™ concentrators. The specific activity of ¹⁵³ Sm labeled IgGthus prepared was about 0.16 μCi/μg of protein. The integrity of the[¹⁵³ Sm(EA-DO3A)]-IgG preparation was verified by HPLC (Sivakoff, S. I.,BioChrom. 1(1), 42-48 (1986)] and standard biochemical procedures, e.g.sodium dodecyl sulfate:polyacrylamide gel electrophoresis andautoradiography, and solid phase radioimmunoassay (RIA). [See DavidColcher et al., Cancer Res. 43, 736-742 (1983).]

EXAMPLE X

Conjugation of1-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid, samarium-153 complex to Fragment F(ab')₂ of CC-49; [¹⁵³Sm(SCN-EA-DO3A)]-Fragment F(ab')₂ of CC-49.

The F(ab')₂ fragment of CC-49, prepared by enzymatic digestion accordingto the procedure described by Lamoyi and Nisonoff [E. Lamoyi and ANisonoff, J. Immunol. Methods, 56, 235-243 (1983)], (225 μl of 1×10⁻⁴ Min 50 mM HEPES, pH 8.5) was mixed with 2.9×10⁻⁸ moles of ¹⁵³Sm(SCN-EA-DO3A) prepared as described in Example IV. Sodium carbonate(1.0M, about 5 μl) was added to bring the pH to 8.9, and the reactionwas continued for about 2.5 hours. Upon termination, the ¹⁵³ Sm labeledfragment was isolated and characterized as described in Example IX.

EXAMPLE XI (A and B)

In vivo localization of [¹⁵³ Sm(EA-DO3A)]-IgG and [¹⁵³Sm(EA-DO3A)]-F(ab')₂.

The utility of the ¹⁵³ Sm(EA-DO3A) labeled IgG (from Example IX) andF(ab')₂ of CC-49 (from Example X) was demonstrated by the uptake of thelabeled materials by human tumor xenograft in athymic mice. Thus femaleathymic (Nu/Nu, CD1 background) mice were inoculated subcutaneously (S.C.) (0.1 ml/source) with the human colon carcinoma cell line, LS-174 T(approximately 1×10⁶ cells/animal). Approximately 2 weeks afterinoculation, each animal was injected via the tail vein with about 2 μCi(15-30 μg) of ¹⁵³ Sm labeled antibody in PBS. The mice were sacrificedat various time intervals. After exsanguination, the tumor and selectedtissues were excised and weighed, and radioactivity was measured in agamma counter. The counts per minute (CPM) of ¹⁵³ Sm in each tissue wasdetermined and expressed as CPM per gram of tissue per injected dosemultiplied by 100 (% injected dose/gram). Results are shown in FIGS.1-14 and Tables IA and IB. The ¹⁵³ Sm(SCN-Bz-DTPA) labeled IgG andF(ab')₂ of CC-49 (from Example ZA) were included in the study forcomparison. Results are shown in Table IC and ID.

EXAMPLE XII

Conjugation ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7-10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex to antibody; [¹⁵³ Sm(SCN-PA-DOTA)]-IgGconjugate.

The antibody used was CC-49, a murine monoclonal IgG that binds to anepitope of TAG-72, a tumor associated antigen. To conjugate, 178 μl ofthe antibody solution (1.26×10⁻⁴ M in 50 mmole HEPES, pH 8.5) was mixedwith 3.4×10⁻⁸ moles ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7-10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium complex (prepared as described in Example VI), followedby addition of a sodium carbonate solution (1.0M, about 17 μl) to raisethe pH to about 8.9. The reaction was allowed to continue for 2 hours atroom temperature. Upon termination, the ¹⁵³ Sm labeled IgG was isolatedby centrifugal gel filtration on Sephadex™ G-25 (2.2 ml) disposablecolumns, and further purified by HPLC on GF-250 column, eluted with acitrate buffer (0.25M, pH 7.4). The fractions which contained thelabeled IgG were pooled, concentrated and exchanged (3 times) into PBSby use of Centricon™ concentrators. The specific activity of the ¹⁵³ Smlabeled IgG thus prepared was about 0.16 μci/μg of protein. Thehomogeneity and integrity of theα-[2-(4-isothiocyanatophenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-10-tetraaceticacid, samarium-153 complex-IgG preparation was verified by HPLC andstandard biochemical procedures as Example IX.

The following Example is an alternative for making labeled antibodyconjugates, which involves first conjugation of the BFC to the antibody,and the subsequent chelation to yield the radionuclide-BFC labeled Ab.

EXAMPLE XIIA

Preparation ofα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid -Ab CC₄₉ conjugate (IgG CC₄₉ -PA-DOTA); and the sebsequentchelation with ¹⁵³ Sm to form ¹⁵³ Sm-labeled antibody (IgG CC₄₉-PA-DOTA-¹⁵³ Sm).

The ¹⁵³ Sm(PA-DOTA) labeled antibody can be prepared by first couplingthe BFC, e.g.α-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (SCN-PA-DOTA), to an antibody at pH 8-9, followed by chelation with¹⁵³ Sm at pH 6 at room temperature for several hours.

In a typical experiment, IgG CC-49 was concentrated and exchanged threetimes into a carbonate buffer (50 mM, Ph 9.1) in a Centricon™Concentrator (30K molecular weight cut off) to leave a solution withantibody concentration greater than 1.5×10⁻⁴ M. To form the conjugate,161 μl of the antibody solution, containing 25×10⁻⁹ mole of IgG CC-49,was mixed with 5 μl of the SCN-PA-DOTA (5 mM concentration in the samecarbonate buffer, prepared by the procedure of Example 18). The mixturewas allowed to stand at room temperature for 5 hours, and terminated byfiltration through the 30K membrane in the Centricon™ Concentrator at5000 rpm on the Sorvall RT centrifuge. The antibody conjugate wasfurther washed with 2 ml of a 0.25M DTPA solution in PBS at pH 7.4 andfive times (2 ml each time) with MES buffer (20 mM, pH 5.8); centrifugedfor 30 min. after each wash. At the end, the PA-DOTA-IgG conjugate wasconcentrated to a minimum volume (about 100 μl) and its integritychecked by HPLC analysis on a GF-250 column. To the purified conjugatewas added a mixture of ¹⁵³ Sm in 0.1N HCl (50μl) and 20 μl of a MESbuffer (1.0M, pH 6), mixed on a vortex mixer, and allowed to stand atroom temperature overnight. Upon termination by centrifugal gelfiltration, the amount of ¹⁵³ Sm incorporated, estimated by HPLCanalysis, was 0.22 BFC-Sm/antibody. That ¹⁵³ Sm was associated with theantibody through chelation with the BFC, which was linked covalently tothe antibody, and not due to non-specific binding was demonstrated bycomparsion with results from the control experiment. In the controlexperiment, IgG CC-49 solution was mixed with ¹⁵³ Sm mixture underidentical conditions. The antibody isolated in a similar manner, had noappreciable amount of ¹⁵³ Sm associated with it. Thus it indicated thatnon-specific binding of Sm-153 by the antibody did not take place underthese conditions.

EXAMPLE XIII

Conjugation ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex to fragment F(ab')₂ of CC-49; [¹⁵³Sm(SCN-PA-DOTA)]-F(ab')₂ fragment.

The F(ab')₂ fragment of CC-49 [prepared by enzymatic digestion accordingto the procedure described by (E. Lamoyi et al., J. Immunol. Methods 56,235-243 (1983)], (225 μl of 1×10⁻⁴ M in 50 mmole HEPES, pH 8.5) wasmixed with 2.9×10⁻⁸ moles ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex prepared as described in Example VI. Sodiumcarbonate (1.0M, about 9 μl) was added to bring the pH to around 8.9,and the reaction was continued for about 2.5 hours. The ¹⁵³ Sm labeledfragment was isolated and characterized as described in Example IX. Thespecific activity was around 0.4 μCi/μg.

EXAMPLE XIV(A and B)

In vivo localization of the conjugate ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex labeled IgG and F(ab')₂ ; [¹⁵³Sm(SCN-PA-DOTA]-IgG and [¹⁵³ Sm(SCN-PA-DOTA)]-F(ab')₂.

The utility of the conjugate ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex labeled IgG and F(ab')₂ of CC-49 (fromExample XII and XIII) was demonstrated by the uptake of the labeledmaterials by human tumor xenograft in athymic mice. The biolocalizationwas determined using the procedure described in Example XI. Results areshown in FIGS. 1 to 7 for IgG and FIGS. 8 to 14 for F(ab')₂, also TablesIIA and IIB.

The conjugate of ¹⁵³ Sm(SCN-Bz-DTPA) with IgG and F(ab')₂ labeledmaterials (prepared from Example ZA) were included in the study forcomparison. (See Tables IC and ID.)

EXAMPLE XV

Conjugation ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex to Antibody; [¹⁵³ Sm(SCN-BA-DOTA)]-IgG.

Whole IgG of CC-49 (174 μl of 1.2×10⁻⁴ M in 0.25M HEPES, pH 8.7) wasmixed with 2.0×10⁻⁸ moles ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 (2.8 mCi) prepared per Example VIII, followed byaddition of a sodium carbonate solution (1.0M, about 2μl) to maintainthe pH about 8.7. The reaction was allowed to continue for 2.5 hours atroom temperature. Upon termination, the ¹⁵³ Sm labeled IgG was isolatedby centrifugal gel filtration on Sephadex™ G-25 (2.2 ml) disposablecolumns, and further purified by HPLC on GF-250 column, eluted with acitrate buffer (0.025M, pH 7.4). The fractions which contained thelabeled IgG were pooled, concentrated and exchanged (3 times) into PBSby use of Centricon™ concentrators. Homogeneity and integrity of thesamarium-153 labled IgG preparation was verified by HPLC and standardbiochemical procedures as described in Example IX.

EXAMPLE XVI

Conjugation ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex to Fragment F(ab')₂ of CC-49; [¹⁵³Sm(SCN-BA-DOTA)]-fragment F(ab')₂ of CC-49.

The F(ab')₂ of CC-49 (84 μl of 2.4×10⁻⁴ M in 0.25M HEPES buffer, pH8.7), prepared by enzymatic digestion according to the proceduredescribed by Lamoyi et al. was mixed with 2.1×10⁻⁸ moles ofα(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex (prepared by the procedure of Example VIII).Sodium carbonate (1.0M, about 2 μl) was added to bring the pH to 8.7,and the reaction was continued for about 2 hours. Upon termination, the¹⁵³ Sm labeled fragment was isolated and characterized as described inExample IX.

EXAMPLE XVII (A and B)

In vivo localization ofα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex labeled IgG (Example XV) and F(ab')2 (ExampleXVI); [¹⁵³ Sm(BA-DOTA)]-IgG and [¹⁵³ Sm(BA-DOTA]-F(ab')₂.

The in vivo study was conducted according to the protocol described inExample XI, and results are shown in FIGS. 1 to 14 and Tables IIIA andIIIB.

EXAMPLE XVIII (A and B)

In vivo localization ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid ¹⁷⁷ Lu-complex labeled IgG and F(ab')₂ ; [¹⁷⁷ Lu(PA-DOTA)]-IgG and[¹⁷⁷ Lu(PA-DOTA)]-F(ab')₂.

The title compounds were prepared and in vivo studies were conductedaccording to protocol described in Examples V, VI, XI, XII and XIII andresults are shown in Tables IVA and IVB.

EXAMPLE XIX (A and B)

In vivo localization ofα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, yttrium-90 complex labeled IgG and F(ab')₂ ; [⁹⁰ Y(PA-DOTA)]-IgGand [⁹⁰ Y(PA-DOTA)]-F(ab')₂.

The titled compounds were prepared and in vivo studies were conductedaccording to protocol described in Example XI with the exception thattissues were digested and counted by liquid scintillation and resultsare shown in Tables VA and VB.

EXAMPLE XX

In vitro pH stability of [¹⁵³ Sm(BFC)]-CC-49-IgG and [¹⁷⁷Lu(BFC)]-CC-49-IgG.

The [¹⁵³ Sm(BFC)]-CC-49-IgG or [¹⁷⁷ Lu(BFC)]-CC-49-IgG was allowed tostand in a 0.2M NaOAc buffer at pH 6.0, 4.0 and 2.8, at roomtemperature, at a protein concentration of about 5×10⁻⁶ M with about 0.5complex/antibody. Samples were withdrawn at certain time intervals andanalyzed by HPLC (GF-250 column) for the dissociation of ¹⁵³ Sm or ¹⁷⁷Lu activity from the protein. In general, the study was carried out forfive days or until 90% of the radioisotopes have become dissociated.Results are expressed as the initial rate of loss of the radioisotopeper day. The results demonstrated the superior stability of ¹⁵³Sm(PA-DOTA), ¹⁷⁷ Lu(PA-DOTA, ¹⁵³ Sm(BA-DOTA), ¹⁵³ Sm(PA-DOTMA) and ¹⁵³Sm(MeO-BA-DOTA), as compared with the standard [¹⁵³ Sm(Bz-DTPA)] complexat acidic pH. Results are shown in the table below.

This greater stability of the complexes in vitro also correlates wellwith the more rapid clearance of ¹⁵³ Sm and ¹⁷⁷ Lu from the body andnon-target tissue (e.g. kidney, liver); see FIGS. 1-34.

    ______________________________________                     % Loss of Isotope/Day                     pH    BFC            Isotope 6.0       4.0 2.8    ______________________________________    Bz-DTPA        .sup.153 Sm                           <2        35  35    PA-DOTA        .sup.153 Sm                           <2        <2  <2    BA-DOTA        .sup.153 Sm                           <2        <2  <2    PA-DOTA        .sup.177 Lu                           <2        <2  <2    MeOBA-DOTA     .sup.153 Sm                           <2        <2  <2    EA-DO3A        .sup.153 Sm                           <2        90  95    PA-DOTMA       .sup.153 Sm                           <2        <2  --    ______________________________________

EXAMPLE XXI (A and B)

In vivo localization ofα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, samarium-153 complex labeled IgG and F(ab')₂ ; [¹⁵³Sm(MeO-BA-DOTA)]-IgG and [¹⁵³ Sm(MeO-BA-DOTA)]-F(ab')₂.

The titled compounds were prepared according to the procedures describedin Examples V, VI, XII and XIII, and the in vivo studies were conductedby the procedures of Example XI. Results are shown in Tables VIA andVIB.

EXAMPLE XXII

Preparation of ¹⁵³ Sm(PA-DOTMA) complex.

To 100 microliters (μl) of ¹⁵³ Sm (0.3×10-3M in 0.01N HCl; 5 mCi) wereadded 6 μl of PA-DOTMA (high R_(f) diastereomer prepared by theprocedure of Example 29; 5 mM in Milli-Q™ water), 20 μl of a MES buffer(1.0M, pH 6) and 1 μl of a HEPES buffer (0.5M, pH 8.7). This solutionwas mixed on a vortex mixer and the final pH of the mixture was about 6.After heating the mixture at 90° C. for 30 min, it was analyzed by HPLCon GF-250 column for degree of complexation, and yields of 90 percent orbetter, were generally obtained. Characterization of the ¹⁵³ Sm-PA-DOTMAcomplex was provided by comparison with the non-radioactive-Sm complex,which had been synthesized and characterized independently as describedin Example 42.

EXAMPLE XXIII

Preparation of ¹⁵³ Sm(SCN-PA-DOTMA).

To the ¹⁵³ SmPA-DOTMA complex prepared in Example XXII and having beencooled for 20 min. at room temperature was added 10 μl of a 1 percentthiophosgene solution in 90 percent acetonitrile-water medium. Themixture was mixed vigorously on a vortex mixer, and allowed to stand atroom temperature for between 5 to 20 min. The reaction took placeinstaneously as monitored by HPLC anaylsis. To purified the activated¹⁵³ Sm complex, it was extracted 3 times (200 μl each) with chloroformand the aqueous layer was passed throught a PRP cartridge (PRPcartridge=Mini-clean™ cartridge from Alltech Associates, Deerfield,Ill.) pretreated with 5 ml of methanol and 5 ml of a MES buffer (20 mM,pH 5.8). After being washed with 5 ml of the MES buffer and 5 ml ofMilli-Q™ water, it was extracted with 900 μl of 90 percent acetontrileto recover approximately 90 percent of the ¹⁵³ Sm activity, with thefirst 100 μl discarded. The mixture was evaporated to dryness underreduced pressure at temperatures below 40° C., and the residue whichcontained mostly the title product was used for conjugation to theantibody.

EXAMPLE XXIV (A and B)

Conjugation of ¹⁵³ Sm(SCN-PA-DOTMA) to IgG and F(ab')₂ CC₄₉.

In general, the antibody was concentrated and exchanged into a carbonatebuffer (pH 9.1 or 9.5, 50 mM) in a Centricon™ concentrator (30Kmolecular weight cut off) to result in a protein concentration of1.5×10⁻⁴ M or greater. To conjugate, a small volume of the carbonatebuffer, that which is required to bring the final protein concentrationto 1.5×10⁻⁴ M, is added to the isothiocyanato derivative of ¹⁵³Sm(PA-DOTMA) (prepared in Example XXIII), followed by the concentratedantibody, of equimolar to the BFC-¹⁵³ Sm complex. It was mixed on avortex mixer and allowed to react at room temperature for 1 hour oruntil 40 to 50 percent of ¹⁵³ Sm become bound to the antibody, as shownby HPLC analysis on a GF-250 column. The labeled antibody was isolatedby two consecutive centrifugal gel filtration on Sephadex™ G-25 columns(2.2 ml). The homgeneity, integrity and immunoreactivity of the labeledantibody was analyzed by HPLC analysis and standard biochemicaltechniques described previously.

EXAMPLE XXV (A and B)

Biodistribution Studies of ¹⁵³ Sm(PA-DOTMA) labeled IgG and F(ab')₂CC₄₉.

The studies were conducted in the similar fashion as described inExample XI. Results are shown in Tables VIIA and VIIB. See FIGS. 15-28.

EXAMPLE XXVI

Preparation of ¹⁷⁷ Lu (PA-DOTMA) Complex.

To 30 μl of ¹⁷⁷ Lu (6×10⁻³ M in 1.1N HCl; 4 mCi) was added 36 μl ofPA-DOTMA (prepared by the procedure of Example 29); 5 mM solution inmilli-Q™ water). The solution was mixed on a vortex mixer, and 115 μl ofa MES buffer (1.0M, pH 6.0) was added to neutralize the solution toabout pH 5.5 to 6. This was heated at 90° C. for 30 minutes to result inbetter than 90% chelation. The mixture was passed through a PRPcartridge pretreated with 5 ml of methanol and 5 ml of a MES buffer (20mM, pH 5.8). It was washed with 5 ml of milli-Q™ waste. The complexextracted in 1000 μl of 90% acetonitrile accounted for 78% of thestarting ¹⁷⁷ Lu activity (discarding the first 100 μl of the extract).

EXAMPLE XXVII

Preparation of ¹⁷⁷ Lu(SCN-PA-DOTMA).

To the ¹⁷⁷ Lu(PA-DOTMA) complex in 90% acetonitrile obtained in ExampleXXVI was added 6 μl of a 10% thiophosgen in 90% acetonitrile. Thissolution was mixed on a vortex mixer, and after 20 minutes, analyzed forthe formation of the isothiocyanate derivative by HPLC. The reaction is,in general, quantitative. Removal of the solvent and the excessthiophosgene was accomplished by evaporation under reduced pressure attemperatures below 40° C. for 1 to 2 hours. The residue was used forconjugation with the antibody.

EXAMPLE XXVIII

Conjugation of ¹⁷⁷ Lu(PA-DOTMA) to IgG CC49.

The antibody IgG CC49 in a carbonate buffer (50 mM, pH 9.1) was mixedwith an equimolar quantity of the isothiocyanato derivative (prepared bythe procedure of from Example XXVII) in the same buffer. The reactionwas continued for 70 min at an antibody concentration of 1.5×10⁻⁴ M, andthe labeled antibody was isolated and characterized according toprocedures described in Example IX.

EXAMPLE XXIX

Long Term Biodistribution Studies of ¹⁷⁷ Lu(PA-DOTMA) and ¹⁷⁷Lu(PA-DOTA) Labeled IgG CC49.

The long term animal test was conducted with the ¹⁷⁷ Lu labeledantibody, taking advantage of its long half-life time (161 hours). Itwas carried out in Balb/c mice over a three week period, according toprotocols described in Example XI. Results are shown in Table VIIIA. SeeFIGS. 29-34.

In the Figures, which plot the data from the following tables, thesymbols used were:

    ______________________________________                                          Bio.    Conjugates      Symbol  Table   Figures                                          Example    ______________________________________    [.sup.153 Sm(EA-DO3A)]-IgG-CC-49                    .increment.                            IA      1-7   IX    [.sup.153 Sm(EA-DO3A)]-F(ab'2)-                    .increment.                            IB       8-14 X    CC-49    [.sup.153 Sm(Bz-DTPA)]-IgG-CC-49                    ο                            IC      1-7   ZA    [.sup.153 Sm(Bz-DTPA)]-F(ab'2)-                    ο                            ID       8-14 ZA    CC-49    [.sup.153 Sm(PA-DOTA)]-IgG-CC-49                    □                            IIA     1-7   XII    [.sup.153 Sm(PA-DOTA)]-F(ab'2)-                    □                            IIB      8-14 XIII    CC-49    [.sup.153 Sm(BA-DOTA)]-IgG-CC-49                                                IIIA    1-7   XV    [.sup.153 Sm(BA-DOTA)]-F(ab+2)-                                                IIIB     8-14 XVI    CC-49    [.sup.153 Sm(PA-DOTMA)]-IgG-CC-49                    ∇                            VIIA    15-21 XXIV    [.sup.153 Sm(PA-DOTMA)]-F(ab'2)-                    ∇                            VIIB    22-28 XXV    CC-49    [.sup.177 Lu(PA-DOTMA)]-IgG-CC-49                                                VIIIA   29-34 XXIX    [.sup.177 Lu(PA-DOTA)]-IgG-CC-49                            VIIIA   29-34 XXIX    ______________________________________

In the accompanying Figures the data represents the following:

    ______________________________________    FIGURE   ORGAN        TITLE    ______________________________________     1       Blood        Biodistribution of .sup.153 Sm-BFC*     2       Liver        Biodistribution of .sup.153 Sm-BFC*     3       Spleen       Biodistribution of .sup.153 Sm-BFC*     4       Kidney       Biodistribution of .sup.153 Sm-BFC*     5       Tumor        Biodistribution of .sup.153 Sm-BFC*     6       Femur        Biodistribution of .sup.153 Sm-BFC*     7       Whole Body   Biodistribution of .sup.153 Sm-BFC*             Retention     8       Blood        Biodistribution of .sup.153 Sm-BFC**     9       Liver        Biodistribution of .sup.153 Sm-BFC**    10       Kidney       Biodistribution of .sup.153 Sm-BFC**    11       Spleen       Biodistribution of .sup.153 Sm-BFC**    12       Tumor        Biodistribution of .sup.153 Sm-BFC**    13       Femur        Biodistribution of .sup.153 Sm-BFC**    14       Whole Body   Biodistribution of .sup.153 Sm-BFC**             Retention    15       Blood        Biodistribution of .sup.153 Sm-BFC*    16       Liver        Biodistribution of .sup.153 Sm-BFC*    17       Spleen       Biodistribution of .sup.153 Sm-BFC*    18       Kidney       Biodistribution of .sup.153 Sm-BFC*    19       Tumor        Biodistribution of .sup.153 Sm-BFC*    20       Femur        Biodistribution of .sup.153 Sm-BFC*    21       Whole Body   Biodistribution of .sup.153 Sm-BFC*             Retention    22       Blood        Biodistribution of .sup.153 Sm-BFC**    23       Liver        Biodistribution of .sup.153 Sm-BFC**    24       Spleen       Biodistribution of .sup.153 Sm-BFC**    25       Kidney       Biodistribution of .sup.153 Sm-BFC**    26       Tumor        Biodistribution of .sup.153 Sm-BFC**    27       Femur        Biodistribution of .sup.153 Sm-BFC**    28       Whole Body   Biodistribution of .sup.153 Sm-BFC**             Retention    29       Blood        Biodistribution of .sup.177 Lu-BFC***    30       Liver        Biodistribution of .sup.177 Lu-BFC***    31       Spleen       Biodistribution of .sup.177 Lu-BFC***    32       Kidney       Biodistribution of .sup.177 Lu-BFC***    33       Femur        Biodistribution of .sup.177 Lu-BFC***    34       Whole Body   Biodistribution of .sup.177 Lu-BFC***             Retention    ______________________________________     *CC49-IgG in nude mice bearing LS174T tumor     **CC49F(ab').sub.2 in nude mice bearing LS174T tumor     ***CC49IgG in balb/c mice

                  TABLE IA    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(EA-D03A)]-    IgG-CC-49    % INJECTED DOSE/GRAM    (n = 5)           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    23.08  2.36   17.25                                2.40 11.32                                          *1.06                                               5.78 *1.69    Liver    6.95   1.28   6.58 0.56 6.58 0.62 7.62 0.98    Spleen   5.17   0.59   5.06 0.96 4.62 0.72 4.92 1.72    Kidney   4.59   0.68   4.01 0.53 3.74 0.76 3.20 0.59    Tumor    11.44  4.13   27.19                                2.12 53.01                                          9.10 59.01                                                    13.23    Femur    --     --     2.58 0.52 2.78 0.20 4.22 0.17    Tumor Wt. g             0.16   0.09   0.20 0.09 0.30 0.17 0.52 0.34    ______________________________________     *n = 4

                  TABLE IB    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(EA-D03A)]-    F(ab').sub.2 CC-49    % INJECTED DOSE/GRAM    (n = 5)    5 hr          24 hr     48 hr      120 hr    Organ   AVG    STD    AVG  STD  AVG  STD   AVG  STD    ______________________________________    Blood   13.36  0.97   1.52 0.42 0.20 0.06  0.05 0.01    Liver   7.13   0.65   8.69 1.3  8.08 0.8   8.11 0.92    Spleen  5.94   1.24   4.96 1.27 4.81 0.99  5.08 1.43    Kidney  41.60  5.60   64.32                               7.88 64.12                                         *12.59                                               37.63                                                    6.57    Tumor   12.95  1.73   16.84                               3.71 11.50                                         4.56  5.53 1.46    Femur   2.74   0.65   2.45 0.26 3.41 0.75  4.99 0.29    Tumor Wt. g            0.25   0.20   0.34 0.25 0.28 0.14  0.44 0.27    ______________________________________     *n = 4

                  TABLE IC    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(Bz-DTPA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    (n = 10)           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    24.97  3.07   16.05                                2.45 12.81                                          2.69 5.84 1.76    Liver    7.74   1.35   6.01 0.95 5.68 1.07 5.43 1.45    Spleen   5.69   1.04   4.86 1.36 4.72 0.81 3.78 0.63    Kidney   4.52   1.04   3.96 0.88 3.81 0.61 3.76 0.31    Tumor    13.90  3.18   42.86                                14.03                                     46.33                                          8.30 61.32                                                    19.80    Femur    2.36   0.12   1.95 0.38 1.97 0.75 2.25 0.50    Whole Body             85.45  4.74   80.16                                3.58 77.36                                          5.32 70.60                                                    3.32    Retention    ______________________________________

                  TABLE ID    ______________________________________    BIODISTRIBUTION of .sup.153 Sm INJECTED AS [.sup.153 Sm(Bz-DTPA)]-    F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    (n = 10)           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    15.03  1.84   2.13 0.48 0.29 0.08 0.04 0.02    Liver    7.07   0.88   7.03 1.24 6.14 0.67 5.80 1.0    Spleen   4.50   0.97   4.22 1.15 3.65 0.62 3.24 0.42    Kidney   46.74  8.69   80.37                                12.76                                     61.42                                          9.85 30.17                                                    5.17    Tumor    15.95  3.88   18.68                                5.10 15.55                                          2.80 6.54 1.14    Femur    2.48   0.24   1.77 0.28 2.48 0.40 2.85 0.35    Whole Body             83.23  4.64   72.17                                6.20 58.89                                          4.40 40.53                                                    2.48    Retention    ______________________________________

                  TABLE IIA    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(PA-DOTA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    (n = 5)           5.5 hr  25 hr     49 hr     121 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    24.65  2.89   16.18                                2.10 13.22                                          1.08 7.34 4.23    Liver    8.25   1.14   5.92 0.41 5.59 0.52 4.16 1.03    Spleen   6.98   1.70   5.05 0.20 4.27 0.52 4.31 1.95    Kidney   4.01   0.86   3.16 0.55 3.44 0.39 3.26 0.57    Tumor    14.31  *2.00  42.81                                8.83 72.59                                          21.53                                               78.36                                                    33.29    Femur    2.69   0.41   2.02 0.32 1.69 0.37 1.44 0.69    Tumor Wt. g             0.27   0.29   0.34.                                0.26 0.16 0.13 0.28 0.15    ______________________________________     *n = 4

                  TABLE IIB    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(PA-DOTA)]-    F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    (n = 5)           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    14.74  0.76   2.25 0.45 0.33 0.08 0.02 *0.01    Liver    7.83   0.82   5.04 *0.32                                     4.48 0.41 1.61 0.26    Spleen   4.80   0.84   3.27 0.45 3.33 0.82 1.41 0.10    Kidney   51.28  5.83   78.78                                8.40 61.91                                          8.17 19.79                                                    5.75    Tumor    17.99  6.53   22.09                                6.07 14.93                                          1.91 5.81 1.21    Femur    2.43   0.17   1.34 0.25 1.17 0.22 0.51 0.18    Tumor Wt. g             0.20   0.08   0.19 0.13 0.29 0.10 0.45 0.23    ______________________________________     *n = 4

                  TABLE IIIA    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(BA-DOTA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    (n = 5)           5.5 hr  24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood   24.89   1.29   17.53                                1.79 13.61                                          0.53 8.68 2.94    Liver   7.53    0.79   5.46 *0.62                                     5.35 *0.40                                               4.26 *0.58    Spleen  5.52    0.61   5.83 1.07 4.78 0.35 3.76 1.28    Kidney  3.84    0.50   3.96 *0.36                                     3.17 *0.57                                               3.49 *0.43    Tumor   14.41   2.96   38.65                                6.45 55.84                                          11.43                                               85.26                                                    25.70    Femur   2.54    0.34   1.81 0.35 2.08 0.30 1.17 0.48    Tumor Wt. g            0.34    0.22   0.13 0.04 0.20 0.06 0.20 0.15    ______________________________________     *n = 4

                  TABLE IIIB    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(BA-DOTA)]-    F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    (n = 5)           5 hr    24 hr     49 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    15.62  *0.86  2.42 0.42 0.28 0.08 0.03 0.01    Liver    7.69   0.71   6.18 0.82 4.22 0.88 1.56 0.25    Spleen   4.93   0.63   3.94 0.52 2.88 *0.36                                               1.43 *0.30    Kidney   50.29  4.99   77.19                                *3.22                                     60.44                                          7.71 24.98                                                    2.67    Tumor    14.58  2.24   24.28                                1.81 17.65                                          3.25 7.39 2.54    Femur    2.13   0.18   1.36 0.15 1.03 0.28 0.68 0.35    Tumor Wt. g             0.17   0.02   0.12 0.07 0.17 0.06 0.12 0.10    ______________________________________     *n = 4

                  TABLE IVA    ______________________________________    BIODISTRIBUTION OF .sup.177 LU INJECTED AS [.sup.177 Lu(PA-DOTA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    (n = 5)           5 hr    24 hr     48 hr     121 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    26.50  4.32   *19.43                                *1.59                                     14.89                                          1.72 11.73                                                    3.14    Liver    7.47   1.34   *6.74                                *0.31                                     5.11 0.41 4.71 0.63    Spleen   6.29   1.56   *5.47                                *1.24                                     4.43 0.31 5.35 1.82    Kidney   3.86   *0.85  *4.13                                *0.45                                     3.13 *0.30                                               3.57 1.15    Tumor    11.94  3.39   *49.03                                *3.21                                     56.36                                          *4.24                                               92.05                                                    *15.32    Femur    2.79   1.08   *2.15                                *0.30                                     2.11 0.39 1.32 *0.19    Tumor Wt. g             0.13   0.09   *0.09                                *0.02                                     0.08 *0.02                                               0.10 *0.04    ______________________________________     *n = 4

                  TABLE IVB    ______________________________________    BIODISTRIBUTION OF .sup.177 LU INJECTED AS [.sup.177 Lu(PA-DOTA)]-    F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    (n = 5)           5 hr    24 hr     48 hr     121 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    12.65  *0.87  *2.40                                *0.18                                     0.47 0.08 0.04 *0.02    Liver    5.39   *0.27  *5.54                                *0.31                                     3.25 0.45 1.30 *0.24    Spleen   4.21   0.95   *3.60                                *0.55                                     2.60 0.74 1.33 0.27    Kidney   38.82  6.42   *74.40                                *6.13                                     48.45                                          6.47 15.88                                                    2.13    Tumor    10.79  2.69   *19.10                                *2.08                                     15.44                                          2.35 4.69 0.78    Femur    2.13   0.83   *1.45                                *0.33                                     0.95 0.43 0.41 *0.25    Tumor Wt. g             0.10   0.03   *0.10                                *0.03                                     0.07 0.06 0.19 0.13    ______________________________________     *n = 4

                  TABLE VA    ______________________________________    BIODISTRIBUTION OF .sup.90 Y INJECTED AS [.sup.90 Y(PA-DOTA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    n = 3         n = 5      n = 5     n = 5    5 hr          24 hr      48 hr     120 hr    Organ   AVG    STD    AVG  STD   AVG  STD  AVG  STD    ______________________________________    Blood   25.15  1.96   18.22                               2.69  15.85                                          *0.81                                               6.62 2.94    Liver   8.25   0.42   7.61 0.77  6.52 0.96 5.68 1.26    Spleen  5.13   0.26   5.50 0.72  5.66 *0.38                                               4.19 0.88    Kidney  3.04   0.58   4.35 1.06  3.66 0.60 2.47 0.33    Tumor   12.45  0.79   44.73                               **1.43                                     68.41                                          *5.48                                               70.32                                                    21.34    Femur   2.05   0.15   1.81 0.54  1.79 *0.11                                               1.04 0.45    ______________________________________     *n = 4     **n = 3

                  TABLE VB    ______________________________________    BIODISTRIBUTION OF .sup.90 Y INJECTED AS [.sup.90 Y(PA-DOTA)]-    F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    n = 5           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    13.78  1.19   2.22 0.46 0.31 *0.03                                               0.04 0.02    Liver    6.98   0.84   4.13 *0.42                                     3.01 0.33 1.56 0.32    Spleen   4.27   0.35   3.44 *0.49                                     2.54 0.71 1.33 0.54    Kidney   44.60  5.52   65.40                                5.92 42.31                                          4.67 12.36                                                    2.60    Tumor    13.08  *0.88  22.44                                4.77 17.84                                          5.09 5.83 2.09    Femur    1.42   0.22   1.12 0.14 0.39 0.18 0.38 0.12    ______________________________________     *n = 4

                  TABLE VIA    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(MeO-BA-    DOTA)]-IgG-CC-49    % INJECTED DOSE/GRAM           n = 5   n = 5     n = 5     n = 4           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    21.00  *0.75  14.57                                1.18 13.98                                          0.78 9.49 1.06    Liver    9.00   1.93   6.22 0.68 6.32 0.41 4.34 0.27    Spleen   5.39   1.40   4.00 0.64 4.69 0.93 4.53 0.41    Kidney   3.48   *0.24  3.23 0.41 3.47 0.45 3.47 0.19    Tumor    18.00  8.91   41.64                                3.37 58.34                                          5.78 69.26                                                    21.92    Femur    2.54   0.27   1.94 0.32 2.05 0.29 1.29 0.16    ______________________________________     *n = 4

                  TABLE VIB    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(MeO-BA-    DOTA)]-F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    n = 5         n = 5     n = 4     n = 5    5 hr          24 hr     48 hr     120 hr    Organ  AVG    STD     AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood  12.98  **0.36  2.13 0.37 0.31 *0.03                                              0.05 0.03    Liver  6.58   **0.16  6.77 1.13 4.62 0.27 2.46 0.55    Spleen 3.95   0.57    3.53 0.76 2.59 0.40 2.18 0.68    Kidney 47.07  5.85    74.16                               8.03 47.68                                         6.29 24.25                                                   2.74    Tumor  12.59  3.96    17.30                               1.68 12.33                                         *0.80                                              5.21 *0.63    Femur  2.61   0.30    1.61 0.31 0.90 0.03 0.68 **0.10    ______________________________________     *n = 3     **n = 4

                  TABLE VIIA    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(PA-    DOTMA)]-IgG-CC-49    % INJECTED DOSE/GRAM    n = 5           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    23.00  1.11   15.58                                1.37 11.78                                          0.36 8.98 0.91    Liver    7.71   0.51   6.68 1.16 4.98 0.32 4.72 0.69    Spleen   6.05   1.03   5.28 0.45 3.66 0.35 3.72 0.53    Kidney   3.68   *0.23  4.07 0.41 3.95 *0.15                                               3.17 0.86    Tumor    10.40  2.10   31.85                                *2.52                                     44.70                                          10.03                                               66.45                                                    13.60    Femur    1.94   0.20   1.66 0.11 1.49 0.10 1.22 0.08    ______________________________________     *n = 4

                  TABLE VIIB    ______________________________________    BIODISTRIBUTION OF .sup.153 Sm INJECTED AS [.sup.153 Sm(PA-    DOTMA)]-F(ab').sub.2 -CC-49    % INJECTED DOSE/GRAM    n = 5           5 hr    24 hr     48 hr     120 hr    Organ    AVG    STD    AVG  STD  AVG  STD  AVG  STD    ______________________________________    Blood    12.85  1.06   1.84 0.13 0.38 0.09 0.07 0.02    Liver    6.12   0.52   5.36 0.49 3.65 0.65 1.93 0.32    Spleen   4.08   0.37   2.88 0.31 2.09 0.62 0.92 *0.13    Kidney   50.28  5.26   58.59                                5.63 45.29                                          7.04 14.85                                                    *0.66    Tumor    11.85  2.23   14.27                                1.02 15.26                                          2.83 4.90 2.01    Femur    1.77   0.06   1.06 0.13 0.96 0.28 0.79 0.24    ______________________________________     *n = 4

                                      TABLE VIIIA    __________________________________________________________________________    24 hr     48 hr 7 day  14 day 21 day    Organ        AVG           STD              AVG                 STD                    AVG                       STD AVG                              STD AVG                                     STD    __________________________________________________________________________    BIODISTRIBUTION OF .sup.177 Lu INJECTED AS [.sup.177 Lu(PA-DOTMA)]-IgG-CC-    49    % INJECTED DOSE/GRAM    n = 5    Blood        31.35           2.66              30.25                 *0.93                    22.47                       0.71                           14.13                              1.05                                  9.88                                     0.83    Liver        10.01           0.46              11.22                 1.47                    8.61                       0.79                           6.76                              0.67                                  5.41                                     1.39    Spleen        10.13           1.16              10.91                 *0.67                    10.79                       *0.36                           8.09                              0.98                                  6.03                                     0.55    Kidney        6.52           2.06              6.14                 0.76                    5.21                       0.97                           3.44                              0.43                                  2.17                                     0.22    Femur        3.97           0.45              4.53                 0.38                    2.84                       0.16                           2.36                              0.23                                  1.67                                     0.30    BIODISTRIBUTION OF .sup.177 Lu INJECTED AS [.sup.177 Lu(PA-DOTA)]-    IgG-CC-49    % INJECTED DOSE/GRAM    n = 5    Blood        30.23           *0.57              28.42                 2.02                    21.96                       1.03                           14.73                              0.48                                  8.85                                     1.03    Liver        11.54           1.34              10.84                 1.27                    9.20                       1.04                           6.69                              0.48                                  6.07                                     1.41    Spleen        10.86           1.47              11.55                 1.64                    10.62                       1.25                           9.25                              1.10                                  6.64                                     1.43    Kidney        7.67           1.83              5.80                 0.59                    4.98                       0.50                           3.27                              *0.19                                  2.60                                     0.64    Femur        3.65           0.62              3.64                 0.51                    2.98                       0.41                           2.50                              0.20                                  1.71                                     *0.17    __________________________________________________________________________     *n = 4

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

What we claim is:
 1. A compound of the formula: ##STR16## wherein: eachQ is independently hydrogen or (CHR⁵)_(p) CO₂ R;Q¹ is hydrogen or(CHR⁵)_(w) CO₂ R; each R independently is hydrogen, benzyl or C₁ -C₄alkyl;with the proviso that at least two of the sum of Q and Q¹ must beother than hydrogen; each R⁵ independently is hydrogen, C₁ -C₄ alkyl or--(C₁ -C₂ alkyl)phenyl; X and Y are each independently hydrogen or maybe taken with an adjacent X and Y to form an additional carbon-carbonbond; n is 0 or 1; m is an integer from 0 to 10 inclusive; p=1 or 2; r=0or 1; w=0 or 1;with the proviso that n is only 1 when X and/or Y form anadditional carbon-carbon bond, and the sum of r and w is 0 or 1; R² isselected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, maleimido,bromoacetamido and carboxyl; R³ is selected from the group consisting ofC₁ -C₄ alkoxy, --OCH₂ CO₂ H, hydroxy and hydrogen; R⁴ is selected fromthe group consisting of hydrogen, nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, maleimido, bromoacetamido andcarboxyl;with the proviso that R² and R⁴ cannot both be hydrogen but oneof R² and R⁴ must be hydrogen; or a pharmaceutically acceptable saltthereof.
 2. A compound of claim 1 wherein the pharmaceuticallyacceptable salt is selected from the group consisting of potassium,sodium, lithium, ammonium, calcium, magnesium, and hydrogen chlorideadducts.
 3. A compound of claim 1 wherein R and R⁵ are both hydrogen. 4.A compound of claim 1 wherein R is hydrogen, R⁵ is methyl and w is
 0. 5.A compound of claim 1 wherein n and r are both 0 and m is 0 through 5.6. A compound of claim 1 wherein R³ and R⁴ are hydrogen.
 7. A compoundof claim 1 having the formula ##STR17## wherein: each Q is independentlyhydrogen or CHR⁵ CO₂ R;each R independently is hydrogen, benzyl or C₁-C₄ alkyl;with the proviso that at least two of Q must be other thanhydrogen; m is an integer from 0 to 5 inclusive; R² is selected from thegroup consisting of hydrogen, nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, maleimido, bromoacetamido andcarboxyl; R³ is selected from the group consisting of C₁ -C₄ alkoxy,--OCH₂ CO₂ H, hydroxy and hydrogen; R⁴ is selected from the groupconsisting of hydrogen, nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; each R^(5')independently is hydrogen or C₁ -C₄ alkyl;with the proviso that R² andR⁴ cannot both be hydrogen but one of R² and R⁴ must be hydrogen; or apharmaceutically acceptable salt thereof.
 8. A compound of claim 7having the formula ##STR18## wherein: each Q is independently hydrogenor CHR⁵ CO₂ R;each R independently is hydrogen, benzyl or C₁ -C₄alkyl;with the proviso that at least two of Q must be other thanhydrogen; m is an integer from 0 to 5 inclusive; R³ is selected from thegroup consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H and hydroxy; R⁴ isselected from the group consisting of nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, maleimido, bromoacetamido andcarboxyl; each R⁵ independently is hydrogen or C₁ -C₄ alkyl; or apharmaceutically acceptable salt thereof.
 9. The compound of claim 8which is1-(2-methoxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.
 10. The compound of claim 8 which is1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.
 11. A compound of claim 6 having the formula ##STR19## wherein:each Q is independently hydrogen or CHR⁵ CO₂ R;Q¹ is hydrogen or(CHR⁵)_(w) CO₂ R; each R independently is hydrogen, benzyl or C₁ -C₄alkyl;with the proviso that at least two of the sum of Q and Q¹ must beother than hydrogen and one of Q is hydrogen; m is an integer from 0 to5 inclusive; w is 0 or 1; R² is selected from the group consisting ofhydrogen, nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; R³ isselected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen; R⁴ is selected from the group consisting ofhydrogen, nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; each R⁵independently is hydrogen or C₁ -C₄ alkyl;with the proviso that R² andR⁴ cannot both be hydrogen but one of R² and R⁴ must be hydrogen; or apharmaceutically acceptable salt thereof.
 12. The compound of claim 11wherein R is hydrogen, R⁵ is methyl and w is
 0. 13. A compound of claim6 having the formula ##STR20## wherein: each Q is independently hydrogenor CHR⁵ CO₂ R;each R independently is hydrogen, benzyl or C₁ -C₄alkyl;with the proviso that at least one Q must be other than hydrogen;m is an integer from 0 to 5 inclusive; R² is selected from the groupconsisting of hydrogen, nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; R³ isselected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,hydroxy and hydrogen; R⁴ is selected from the group consisting ofhydrogen, nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; each R⁵independently is hydrogen or C₁ -C₄ alkyl;with the proviso that R² andR⁴ cannot both be hydrogen but one of R² and R⁴ must be hydrogen; or apharmaceutically acceptable salt thereof.
 14. A compound of claim 13having the formula ##STR21## wherein: each Q is independently hydrogenor CHR⁵ CO₂ R;each R independently is hydrogen, benzyl or C₁ -C₄alkyl;with the proviso that at least one Q must be other than hydrogen;m is an integer from 0 to 5 inclusive; R² is selected from the groupconsisting of nitro, amino, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido and carboxyl; each R⁵independently is H or C₁ -C₄ alkyl; or a pharmaceutically acceptablesalt thereof.
 15. The compound of claim 14 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic4,7,10-tris-(R-methylacetic) acid, 1-isopropyl-4,7,10-trimethyl ester.16. The compound of claim 14 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic4,7,10-tris-(R-methylacetic) acid.
 17. The compound of claim 14 which isα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic)acid.
 18. The compound of claim 14 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid, mixed ammonium, potassium salt.
 19. The compound of claim 14 whichis α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid.
 20. The compound of claim 14 which isα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid.21. The compound of claim 14 which isα-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 22. The compound of claim 14 which isα-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 23. The compound of claim 14 which isα-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester.
 24. The compound of claim 14 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, 1,4,7,10-tetramethyl ester.
 25. The compound of claim 14 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 26. The compound of claim 14 which isα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 27. A compound of claim 1 having the formula ##STR22## wherein:each Q is independently hydrogen or CHR⁵ CO₂ R;each R independently ishydrogen, benzyl or C₁ -C₄ alkyl;with the proviso that at least one Qmust be other than hydrogen; m is an integer from 0 to 5 inclusive; R³is selected from the group consisting of C₁ -C₄ alkoxy, --OCH₂ CO₂ H,and hydroxy; R⁴ is selected from the group consisting of nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, maleimido,bromoacetamido and carboxyl; each R⁵ independently is hydrogen or C₁ -C₄alkyl; or a pharmaceutically acceptable salt thereof.
 28. The compoundof claim 27 which isα-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 29. The compound of claim 27 which isα-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, tetraamonium salt.
 30. The compound of claim 27 which isα-(2-methoxy-5-isothiocynatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid, tetraamonium salt.
 31. A complex which comprises a compound of theformula ##STR23## wherein: each Q is independently hydrogen or(CHR^(5'))_(p) CO₂ H;Q¹ is hydrogen or (CHR^(5'))_(w) CO₂ H;with theproviso that at least two of the sum of Q and Q¹ must be other thanhydrogen; each R^(5') independently is hydrogen, C₁ -C₄ alkyl or --(C₁-C₂ alkyl)phenyl; X and Y are each independently hydrogen or may betaken with an adjacent X and Y to form an additional carbon-carbon bond;n is 0 or 1; m is an integer from 0 to 10 inclusive; p=1 or 2; r=0 or 1;w=0 or 1;with the proviso that n is only 1 when X and/or Y form anadditional carbon-carbon bond, and the sum of r and w is 0 or 1; R² isselected from the group consisting of hydrogen, nitro, amino,isothiocyanato, semicarbazido, thiosemicarbazido, maleimido,bromoacetamido and carboxyl; R³ is selected from the group consisting ofC₁ 14 C₄ alkoxy, --OCH₂ CO₂ H, hydroxy and hydrogen; R⁴ is selected fromthe group consisting of hydrogen, nitro, amino, isothiocyanato,semicarbazido, thiosemicarbazido, maleimido, bromoacetamido andcarboxyl;with the proviso that R² and R⁴ cannot both be hydrogen but oneof R² and R⁴ must be hydrogen; or a pharmaceutically acceptable saltthereof;complexed with an ion of a metal selected from the groupconsisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb,Lu, Y and Sc.
 32. A complex of claim 31 wherein the metal ion isselected from the group consisting of Sm, Ho, Lu and Y.
 33. A complex ofclaim 31 wherein the metal ion is selected from the group consisting of¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ¹⁴⁹ Pm, ¹⁵⁹ Gd, ¹⁴⁰ La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ Sc and¹⁴² Pr.
 34. A complex of claim 33 wherein the metal ion is selected fromthe group consisting of ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu and ⁹⁰ Y.
 35. A complexof claim 31 wherein the metal ion is complexed with the compound ofclaim
 1. 36. A complex of claim 33 wherein the metal ion is complexedwith the compound of claim
 1. 37. A complex of claim 31 wherein themetal ion is complexed with the compound of claim
 7. 38. A complex ofclaim 33 wherein the metal ion is complexed with the compound of claim7.
 39. A complex of claim 31 wherein the metal ion is complexed with thecompound of claim
 8. 40. A complex of claim 33 wherein the metal ion iscomplexed with the compound of claim
 8. 41. A complex of claim 31wherein the metal ion is complexed with the compound of claim
 11. 42. Acomplex of claim 32 wherein the metal ion is complexed with the compoundof claim
 11. 43. A complex of claim 31 wherein the metal ion iscomplexed with the compound of claim
 13. 44. A complex of claim 33wherein the metal ion is complexed with the compound of claim
 13. 45. Acomplex of claim 31 wherein the metal ion is complexed with the compoundof claim
 14. 46. A complex of claim 33 wherein the metal ion iscomplexed with the compound of claim
 14. 47. A complex of claim 31wherein the metal ion is complexed with the compound of claim
 27. 48. Acomplex of claim 33 wherein the metal ion is complexed with the compoundof claim
 27. 49. A complex of claim 33 wherein the metal ion iscomplexed with the compound of claim
 21. 50. A complex of claim 49wherein the metal ion is ¹⁵³ Sm complexed with the compound of claim 21.51. A complex of claim 49 wherein the metal ion is ⁹⁰ Y complexed withthe compound of claim
 21. 52. A complex of claim 33 wherein the metalion is complexed with the compound of claim
 22. 53. A complex of claim52 wherein the metal ion is ¹⁵³ Sm complexed with the compound of claim22.
 54. A complex of claim 33 wherein the metal ion is complexed withthe compound of claim
 25. 55. A complex of claim 54 wherein the metalion is ¹⁵³ Sm complexed with the compound of claim
 25. 56. A complex ofclaim 54 wherein the metal ion is ⁹⁰ Y complexed with the compound ofclaim
 25. 57. A complex of claim 54 wherein the metal ion is ¹⁷⁷ Lucomplexed with the compound of claim
 25. 58. A complex of claim 54wherein the metal ion is ¹⁶⁶ Ho complexed with the compound of claim 25.59. A complex of claim 33 wherein the metal ion is complexed with thecompound of claim
 26. 60. A complex of claim 59 wherein the metal ion is¹⁵³ Sm complexed with the compound of claim
 26. 61. A complex of claim59 wherein the metal ion is ⁹⁰ Y complexed with the compound of claim26.
 62. A complex of claim 59 wherein the metal ion is ¹⁷⁷ Lu complexedwith the compound of claim
 26. 63. A complex of claim 59 wherein themetal ion is ¹⁶⁶ Ho complexed with the compound of claim
 26. 64. Acomplex of claim 33 wherein the metal ion is complexed with the compoundof claim
 29. 65. A complex of claim 64 wherein the metal ion is ¹⁵³ Smcomplexed with the compound of claim
 29. 66. A complex of claim 33wherein the metal ion is complexed with the compound of claim
 30. 67. Acomplex of claim 66 wherein the metal ion is ¹⁵³ Sm complexed with thecompound of claim
 30. 68. A complex of claim 33 wherein the metal ion iscomplexed with the compound of claim
 16. 69. A complex of claim 68wherein the metal ion is ¹⁵³ Sm complexed with the compound of claim 16.70. A complex of claim 68 wherein the metal ion is ⁹⁰ Y complexed withthe compound of claim
 16. 71. A complex of claim 68 wherein the metalion is ¹⁷⁷ Lu complexed with the compound of claim
 16. 72. A complex ofclaim 68 wherein the metal ion is ¹⁶⁶ Ho complexed with the compound ofclaim
 16. 73. A complex of claim 33 wherein the metal ion is complexedwith the compound of claim
 17. 74. A complex of claim 73 wherein themetal ion is ¹⁵³ Sm complexed with the compound of claim
 17. 75. Acomplex of claim 73 wherein the metal ion is ⁹⁰ Y complexed with thecompound of claim
 17. 76. A complex of claim 73 wherein the metal ion is¹⁷⁷ Lu complexed with the compound of claim
 17. 77. A complex of claim73 wherein the metal ion is ¹⁶⁶ Ho complexed with the compound of claim17.
 78. The complex of claim 31 which is1-(2-methoxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.
 79. The complex of claim 31 which is1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid.
 80. The complex of claim 31 which isα-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.
 81. The complex of claim 31 which isα-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid.